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HID EVOlution™ - qPCR/STR Setup System Getting Started Guide

1. Figure 7 STR PCR plate layout for the AmpF STR Yfiler kit D007 Amplification positive control control DNA 007 D9974A Amplification positive control control DNA 9947A NTC Amplification negative control TE buffer LDR empty wells indicating where allelic ladder sample replicates will be placed in the CE plate The positive and negative control volumes are 10 ul for 25 uL reactions and 20 uL for 50 pL reactions For more information For information on Reports see the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 6 Results Running scripts and on script error messages see the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 2 Running a STR PCR Setup Script and Section 8 4 Application Software Freedom EVOware software see the Tecan EVOware Standard EVOware Plus 2 1 Software Manual and Tecan EVOware Standard EVOware Plus 2 1 Software Getting Started Guide HID EVOlution gPCR STR Setup System Getting Started Guide 63 7 Chapter 7 Run Automated STR PCR Setup For more information 64 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis This chapter explains where to get relevant information to perform STR PCR and how The HID EVOlution fO Set up for CE
2. Phone eil Manual Manual Automated Automated Percent Difference ng uL RFU Average RFU Average Manual vs Automated 0 025 250 66 1074 128 250 99 1005 63 10096 0 05 561 20 493 50 11496 0 1 852 30 985 80 8696 0 5 986 70 873 00 11396 1 1160 60 977 40 11996 2 1125 70 1000 50 11396 5 1224 80 1061 80 11596 10 1025 00 1072 50 9696 25 1008 30 1041 90 9796 50 1211 30 1033 40 11796 HID EVOlution qPCR STR Setup System Getting Started Guide 123 E Appendix E Validation Experiments and Results Identifiler and SGM Plus STR PCR amplification reaction setup accuracy study Figure 38 below and Table 21 on page 125 show data from the SGM Plus kit STR PCR setup The STR PCR setup was run using a 50 uL PCR reaction script The peak height variation is analogous to the data generated from the manually prepared samples The combined average peak height for all samples 2 0 1 ng uL was 983 RFU 80 for the manual setup and 1066 RFU 174 for the automated setup Correlation Between Manual and Automated Operation 3000 4 25004 2000 4 1500 4 Average Peak Height 1000 4 T 0 il T Source DNA ng uL Figure 38 Average peak heights at varying starting DNA concentrations Results from SGM Plus PCR amplification reactions prepared by the HID EVOlution system are shown in yellow those prepared manually are shown in green Positiv
3. 3500 i Plate Tube 3000 d Lu c v 2500 l IE 4 o Q7 Ee x 2000 H EN TEF x U il E 1500 z s S 1000 z TM 0 T T T 0 025 0 1 0 5 1 2 5 10 50 Source DNA ng uL Figure 28 SGM Plus kit reproducibility studies depicting the average peak height distribution of each study Samples were either in a plate brown or in a tube green Studies were carried out on three separate days In conclusion sample dilution and normalization including the direct transfer for automated SGM Plus kit samples was successful regardless of the DNA starting concentration and source vessel HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Precision and reproducibility studies STR PCR amplification reaction setup scripts Other AmpF STR kits Supplemental precision and reproducibility studies The remaining data from the precision and reproducibility study of the STR PCR amplification setup script validation are shown in the following figures sample data from the Identifiler and SGM Plus kits were detailed in the preceding sections Representative samples of STR profiles are also included Precision studies for Profiler Plus kit peak height averages Precision studies for COfiler kit peak height averages Precision studies for SEfiler kit peak height
4. Table8 Identifiler kit reagent volumes 25 pL reaction volume for STR PCR amplification Required Extracted Control Required Required Available Volume DNA Excess Dead Samples 8 Reagent Volume in per Samples er Run Reactions Volume u 9 Full Tube Reaction per Run p per Run per Tube Minimum Required Volume for of Reagent 88 Samples and 2 Controls A B c D E AmpF STR Primer Set 1 1 mL 6 05 uL up to 88 2 4 50 uL A x B C D E 619 uL AmpF STR PCR Reaction Mix 1 1 mL 11 55 uL up to 88 2 4 50 uL Ax B C D E 1136 uL in one tube AmpliTag Gold DNA Polymerase 50 uL 100 uL combined in one tube for 1 to 88 samples plus 2 controls 100 pL combined in one tube AmpF STR Control DNA 9947A 300 uL 60 uL for 1 to 88 samples plus 2 controls 60 uL T4o9Eo 4 buffer in reagent block NA 60 uL for 1 to 88 samples plus 2 controls 60 uL T4o9Eo 4 buffer in trough NA 25 mL in trough for 1 to 88 samples plus 2 controls 25 mL in trough t Includes excess volume to compensate for evaporation and pipetting losses during the run A 50 pL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips 6 05 uL reaction x 88 2 4 reactions 50 uL tube 618 7 uL in one tube tt 11 55 ul reaction x 88 2 4 reactions 50 uL tube 1135 7 uL in one tube If you divide the minimum volume of PCR Reaction Mix between two tu
5. 5000 B Plate Bl Tube 4000 4 i i o9 3000 s 5 o9 a amp 2000 gt X 1000 i i E B T T T T T T T T T 0 025 0 1 0 5 1 2 5 10 50 Positive Source DNA ng uL Figure 31 Precision studies for the SEfiler kit Figures 32 and Figure 33 on page 119 show peak height averages across different starting concentrations Male Control refers to the positive control used in the experiment Green bars represent sample data processed from a plate P and yellow bars represent sample data processed from tubes T the source vessels 118 HID EVOlution gPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Precision and reproducibility studies STR PCR amplification reaction setup scripts Yfiler Plate and Tube Study 8000 7000 4 2 E I H T T T T T T T 6000 x bk k 5000 4 4000 l 3000 4 20004 1000 4 04 Peak Height E T T T T T T T T T CEU NC NT UC WA ne PAD aA ae eon 2 Z 4 2 d d NN 4 V o o NO e AS AS n gt AS es o S S d Y 9 lt O OJQ 9 Source DNA ng pL Figure 32 Precision studies for the Yfiler kit MiniFiler Plate and Tube Study 7000 lt z 6000 Ps 5000 9 x a x X 4000 9 a 9 3000 5 2000 i E 1000 04 SERE E EEG NUN EU e
6. 0 eee x The HID EVOlution qPCR STR Setup System 1 HID EVOlution gPCR STR Setup System workflow 00 0 cece cece eee eee 2 Supported system configuration 2 0 00 eee 4 supported kits moe PIRE EA P ee ea aa eee es 5 Required instruments software and materials 0 0 0 0 eee eee 6 For more information lisse 7 Pre Run Procedures sauces Eo aw RES Eea ENa RA AE 9 One time tasKs vx EE Sx aer esce anser oss a xU tA ERR E RS RE ES EORR 10 Before each run Run maintenance scripts 000 00 e eee eee 12 Before each run Set up extracted DNA samples lllllll selle 13 Before each run Optional Set up sample information eee ees 17 Guidelines Preventing contamination llis 20 For more information 0 rira idar dana ehh 21 Prepare qPCR Reagents and Labware sss 23 Review pre run checklist for qPCR reaction setup 20 000 eee ee 24 Set UP reagents nese ese tke Seek dee Sie eund Ge he ek hla ag bade S 25 Set up the labware on the worktable 0 0 0 eee 30 Run Automated qPCR Setup 22 22000000e ee enee 33 Run a qPCR setup script 0 0000 cee eee 34 Perform post run taSkS oa ssaa ani a E aa E a E a aa Ea ALa eee 35 About the qPCR reaction plate layout 0 2 0 ee eee 36 For more information 0 000 eee 37 Contents Chapter 5 Chapter 6 Chapter
7. Available volume in full Minimum volumet Reagent tube of reagent required on worktable Undiluted Quantifiler Human DNA Standard 120 uL 100 uL T4 Eo buffer in trough 6 mL in trough To prepare a reaction plate using EITHER the Quantifiler NA Human or Quantifiler Y Human Male Quantification Kit To prepare a reaction plate with BOTH the Quantifiler NA Human and Quantifiler Y Human Male Quantification Kits 7 mL in trough t Includes 50 uL dead volume per tube and 5 mL per trough necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips 3 Calculate the required volume of Quantifiler kit reagents using the Table 6 or Table 7 on page 28 IMPORTANT To obtain the required volume of primer mix you can place up to 3 tubes of primer mix in the reagent block for one run If you divide the minimum required volume of primer mix across 2 or 3 tubes each tube must contain a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions See About minimum required reagent volumes on page 25 HID EVOlution qPCR STR Setup System Getting Started Guide 27 N apInd peuejs Buje wayshs dnies H LS HOdb uonniOAd AIH Table 6 Reagent volumes for qPCR reaction setup with either the Quantifiler Human or Y Human Male Quantification Kit
8. Applied Getting Started Guide KS Biosystems A Getting Started Guide E BP seins HID EVOlution qPCR STR Setup System Getting Started Guide For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT AL LOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER OF QUANTIFILER KITS LIMITED LICENSE Use of this product including the Quantifiler Duo DNA Quantification Kit is covered by US patent claims and patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in forensic and paternity testing including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal rese
9. fo lt 1 2 i memm O 0 1 E 0 01 T T T T T 0 025 0 05 0 1 1 0 5 0 25 DNA Source ng uL Figure 25 Precision study to verify Quantifiler Y Human Male kit script plate top and tube bottom n 1 2 Combined Quantifiler Human and Quantifiler Y Human Male reaction setup precision study Experiment The HID EVOlution qPCR scripts either Quantifiler Human Y plate esc or Quantifiler Human Y tubes esc which run both the Quantifiler Human and Y Human Male assays on the same plate were tested for assay setup precision using different quantities of human DNA on the same 96 well optical plate Samples containing about 0 025 0 05 0 1 1 0 5 0 and 25 0 ng uL of DNA were quantified for 12 replicates in three runs HID EVOlution gPCR STR Setup System Getting Started Guide 111 E Appendix E Validation Experiments and Results Precision and reproducibility studies qPCR reaction setup scripts Figure 26 shows a box and whisker plot of the average DNA quantity for each input amount and quantification kit The red boxes represent samples run with Quantifiler Human Quantification kit reagents and the green boxes represent DNA samples run with the Quantifiler Y Human Male Quantification kit reagents Quantifiler Script Verification Plate 100 Kits i H_Plate a 2 Hi Y Plate 2 10 a t 1 7 a 3 o lt x EJ 0 1 E m u 0 01 Kits KR Fi E V A m a E E
10. 100 100 100 100 100 100 100 100 100 100 100 100 Positive Negative ladder 100 ContainerType sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Allelic 96 we11 GeneMapper Gener i C I nstance Comment Priority None None None None None None None None None None None None 100 100 Ladder AppType Owner Regu Sample Type HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced HID_Advanced Positive Control Negative Control HID Advanced None HID EVOlution qPCR STR Setup System Getting Started Guide ar No log in Operator NO log in Snp Set Analysis Method Panel Identifiler_v1 Identifiler v1 Identifiler v1 Identifiler v1 Identifiler v1 Identifiler vi Identifiler v1 Identifiler v1 Identifiler vi Identifiler v1 Identifiler v1 Identifiler v1 None HID Advanced None HID Advanced Identifiler v1 User Ider Ider 67 Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis Transfer and import the CE Setup file Transfer and import the CE Setup file IMPORTANT To successfully import the CE Setup file to the 3130 3130x Genetic Analyzer Data Collection software v3 0 the instrument protocol and results group names created in the Data Collection software must match those in the CE Setup file See Create an instr
11. _Sng uL 0 1ng uL Wr il no Lu dl Wo en gd 9 92 3 3 3 5 Oo ww 8 6 8 8 2 55352557 535 2ng uL 0 025ng uL 1 bi Li ETENIM Figure 36 COfiler kit STR profiles HID EVOlution qPCR STR Setup System Getting Started Guide 121 Appendix E Validation Experiments and Results Identifiler and SGM Plus STR PCR amplification reaction setup accuracy study Identifiler and SGM Plus STR PCR amplification reaction setup accuracy study 122 Experiment Results The AmpF STR kits were grouped based on the PCR reaction volume 25 uL for Identifiler Yfiler and MiniFiler kits 50 uL for SGM Plus Profiler Plus COfiler and SEfiler kits Each script was verified for functionality One kit specific script from each group was selected to test for further validation Identifiler and SGM Plus kits The accuracy studies were performed using 0 025 0 1 0 5 1 2 5 10 and 50 ng uL DNA samples that were quantified using the Quantifiler Human kit on the HID EVOlution system Eleven replicates of each DNA concentration were prepared and placed in either a plate or in tubes The HID EVOlution system was then used to normalize the DNA concentration and prepare the STR PCR amplification reactions The experiment was repeated three times for extracted DNA in tubes and three additional times for extracted DNA
12. Normalize the DNA Dilute samples according to the exact concentration determined for each individual samples sample For the Tecan Freedom EVO series DNA dilution protocols and a description of dilution ratios volumes of sample used and volumes of TE used for dilution see Appendix D Dilution Protocols on page 93 or the Tecan HID EVOlution Application Guide Automation for Applied Biosystems Human Identification Kits Section 10 Appendix C HID EVOlution qPCR STR Setup System Getting Started Guide 137 Appendix F Automation Guidelines STR PCR reaction setup automation guidelines Prepare the For instruments other than the Tecan Freedom EVO series use the description below to construct a dilution protocol within the parameters of your specific instrument e Add TE buffer to the predilution plate then dilute the first dilution step samples in well X For a two step dilution dilute with a second step in well X 8 Mix thoroughly in all steps to assure a homogeneous solution Vary the volumes for the TE buffer and the volume of sample transferred from the tube for dilutions of 1 23 to 1 4000 Single step dilutions require a dilution ratio between 1 1 to 1 22 9 while two step dilutions require a higher dilution ratio Use the minimum recommended final volume recommended by the instrument manufacturer for an individual dilution to ensure liquid handling precision IMPORTANT If the concentration of the original cont
13. the sample concentrations were normalized to 0 1 ng uL by the HID EVOlution system and 10 uL of normalized sample was transferred to the PCR reaction plate 120 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Precision and reproducibility studies STR PCR amplification reaction setup scripts Tsongu O Ub o0 uaa MEI g etoal War Md r HI d w o 9 V O9 9 9 9 R MN 39 2 D 2 2 DP B n S 6 9 6 0 8 68 6 8 88 89 2 9 2 8 59 ee lOng uL 0 5ng uL jJ n EE HE ara IE nu HII d co 9 9 9 9 3 9 9 n ewwd 0 950093 6 8 8 9 2 8 555 8B a M 0 M P aH S a n _ Sng uL O 1ng uL 5 uM aw a d D GL MEE I d go wn a5 0 n un 5 7 9 9 V 9S 9 9 9 9 B3 9 53 P 5 5 9 2 5h B MM M s M ir M MM M M M M 2ng uL 0 025ng uL 310 p lll dI dh 7 i ee E Figure 35 Profiler Plus kit STR profiles 1 a E a aug usu 8 8 8 88 S BS 8S 8 2B 8 8S Ma 2 UA HA i AA u A E SOng ul Ing uL ET L doa ora E T EU hl 8e 9 E a 8 8 8 8 BB 2 2 eS 822 8 2 2 AM I Lil 9 9 V 9 9 9 9 3 9 53 n 0 n9 8 S19 0 8 8 8 2 2 82 95 BB S eS Oe 10ng uL O 5ng uL bu Lu luu b ao n Nw UO S 8 8 NM NMNMN BOB ee MA
14. 0 0 aeaee 132 Conclusion baee bee e add duet bbs Died Dados Dioni a 133 OG IET 134 HID EVOlution qPCR STR Setup System Getting Started Guide 103 Appendix E Validation Experiments and Results Overview Overview 104 Importance of validation This appendix describes the experiments performed by the Applied Biosystems Human Identification HID team to validate automated qPCR STR reaction setup on the Tecan HID EVOlution qPCR STR Setup System using Applied Biosystems chemistries instruments and software The experimental design followed the validation guidelines published by the Scientific Working Group on DNA Analysis Methods SWGDAM Validation experiments were carried out to assess the functionality of the HID EVOlution scripts and to assess the overall assay precision accuracy reproducibility and potential for cross contamination in qPCR STR setup experiments automated by the instrument The experimental results were compared to data generated from manually prepared samples IMPORTANT Laboratories should conduct their own validation studies with their own specific workflow and instruments to set their own standards for internal use The HID EVOlution system was tested using the HID EVOlution Software v1 0 the Freedom EVOware Software v1 4 and the Tecan Freedom EVO 150 Liquid Handling Workstation with a 4 channel LiHa a PosID 3 barcode reader and carriers and reagent blocks that were arran
15. 1 920 1 17 1 2 34 37 66 0 0 20 1 920 2 400 1 21 7 2 76 57 24 0 0 20 2 400 3 000 1 26 7 2 25 57 75 0 0 20 3 000 3 692 1 34 5 2 32 77 68 0 0 20 3 692 4 593 1 40 2 78 0 0 20 4 593 6 194 1 52 2 78 10 34 29 66 20 6 194 8 348 1 72 6 2 78 18 00 22 00 20 8 348 11 23 1 96 2 78 23 66 16 34 20 11 23 15 12 1 132 2 78 27 84 12 16 20 15 12 20 43 1 176 2 78 31 00 9 00 20 20 43 27 43 1 240 2 78 33 34 6 66 20 27 43 36 23 1 320 2 78 35 00 5 00 20 36 23 48 00 1 416 2 78 36 16 3 84 20 48 00 64 00 1 564 2 78 37 16 2 84 20 64 00 83 48 1 740 2 78 37 84 2 16 20 83 48 109 71 1 964 2 78 57 51 2 49 20 109 71 142 22 1 1280 2 158 37 50 2 50 20 142 22 182 86 1 1600 2 158 38 00 2 00 20 182 86 240 00 1 2136 2 158 57 75 2 25 20 240 00 295 38 1 2760 2 158 77 68 2 32 20 295 38 355 56 1 3200 2 158 78 2 20 355 56 457 14 1 4000 2 98 158 2 20 HID EVOlution gPCR STR Setup System Getting Started Guide 101 Appendix D Dilution Protocols Dilution protocols with examples Table 16 Dilution protocols for 20 pL transfer Columns 1 and 2 apply only for a DNA target input amount of 1 ng continued If your sample concentration is First Dilution Mixture D1 Second Dilution Mixture D2 Volume between Then the D1 or D2 dilution added to ratio is Volume of Volume from SIRPCR Min Max t extracted TE added to D1 added to TE added to reaction ng pL ng pL DNA added D1 uL D2 uL D2 uL plate uL to D1 uL H 457 14 600 00 1 53
16. 10 e vas wilt ABPTPISIICSEIPIM e 19 28 se 42 so se eo 74 2 o e s roo 27 95 e 51 59 ev s o or o r2 20 29 26 44 52 so eo vo se e 5 13 21 29 sr 45 so ex eo 77 95 99 P e 14 22 20 o o 54 82 70 vo e 7 15 23 9 so 7 ss eo i 79 v os 4 e ne 22 22 40 4o 5o e 72 20 09 98 IMPORTANT Confirm that The reaction plate is placed in the metal plate adapter The reaction plate wells are aligned with the holes in the metal plate adapter e Well AI is positioned in the upper left corner HID EVOlution qPCR STR Setup System Getting Started Guide 31 3 Chapter 3 Prepare qPCR Reagents and Labware Set up the labware on the worktable 000000000000 000000000000 OOo oo oo 00000000 Eo ood Site number 1 O0000000 O0000000 Oo0000000 Oo0000000 00000000 ooog oon Site number 2 Site number 3 oooo oooo oooo oooo oooo oooo oooo oooo 1 82 83 84 85 Grid numbers 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 2 Tecan Freedom EVO workstation worktable layout for a qPCR setup run Locations for DNA samples in both a plate and tubes are shown Note Locations for DNA samples in both plate and tubes are shown but only one type of plasticware for extracted DNA can be placed on the
17. 10096 comparison was observed In conclusion the HID EVOlution Software tracked the sample name and position correctly during processing HID EVOlution qPCR STR Setup System Getting Started Guide 131 Appendix E Validation Experiments and Results Contamination study Contamination study 132 Experiment Results The potential for contamination in the automated quantification and STR protocols including normalization was evaluated Samples that contained 100 uL of 100 ng uL of DNA were alternated with samples that contained TE buffer only On each 96 well plate 40 replicates of the DNA and TE buffer were set up in a checkerboard or vertical stripe pattern These plates served as source plates for a qPCR reaction that used the Quantifiler Human kit and script The negative samples were evaluated for a Cy value lt 40 If a negative sample had a Cy value lt 40 it was evaluated manually for an STR profile using the MiniFiler kit A separate set of similarly patterned source plates were used to test the STR PCR setup operations Samples were processed for quantification using the Quantifiler Human kit diluted to normalize DNA concentrations and then prepared for STR PCR amplification using the Identifiler kit All TE wells in the striped pattern layout exhibited Cy values gt 40 indicating that there was no cross contamination during the qPCR reaction setup Of the 40 TE wells in the checkerboard layout one well ex
18. 12 MicroAmp Optical 96 Well Reaction Plate STR PCR reaction plate with 96 well metal plate adapter 13 Tube racks S1 through S6 for DNA sample tubes if samples are in tubes 56 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 7 Run Automated STR PCR Setup This chapter provides procedures for performing a STR PCR reaction setup run The HID EVOlution E Run STR PCR Setup sop ete ce che cs doe epee deo b Rs Mac ds 58 qPCR STR Setup System E Perform post run tasks 1 2 0 cette teens 61 Take care of the STR PCR reaction plate llle 61 Clean up the instrument 0 eee 61 ErssB h Procedures B About the STR PCR plate layout 0 0 0 es 62 E For more information sss e 63 Prepare qPCR Reagents and Labware Run Automated qPCR Setup Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Chapter 7 Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution qPCR STR Setup System Getting Started Guide 57 ie Chapter 7 Run Automated STR PCR Setup Run STR PCR setup Run STR PCR setup After performing all tasks including maintenance in the Review pre run checklist for STR PCR reaction setup on page 46 use the following procedure to begin a STR PCR reaction setup run 1 Select the appropriate EVOware software script for the AmpF
19. Biosystems 4 and 5 dye STR amplification kits Generate Samples Reports and importable 7500 Setup and CE Setup files appropriate to each kit instrument and software configuration HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Materials and methods Materials and methods Samples Reagents Instrumentation consumables and software Genomic DNA samples obtained from Biochain Hayward CA Sigma Chemical Company St Louis MO and Serological Research Institute Richmond CA were used in all studies DNA standard dilution series were made using the Quantifiler Human DNA Standard provided in the kit being tested A single lot of each kit was used during the validation studies TE buffer containing 0 1 EDTA was purchased from Teknova Hollister CA Applied Biosystems POP 4 PN 4316355 GeneScan 500 LIZ Size Standard PN4322682 GeneScan 500 ROX Size Standard PN 403039 for 50 uL reactions Hi Di Formamide PN 4311320 and Running Buffer 1X prepared from 310 Running Buffer 10X PN 402824 were used for the CE run The Tecan Freedom EVO 150 Liquid Handling Workstation which is equipped with a 4 channel liquid handling arm was used to set up the qPCR reactions normalize DNA sample concentration and to set up STR PCR reactions for STR analysis using the HID EVOlution Software v1 0 the Freedom EVOware Software v1 4 SP1 the Tecan H
20. Concentrate the sample then repeat the test with the respective Quantifiler kit before performing STR analysis HID EVOlution qPCR STR Setup System Getting Started Guide 71 Appendix A Troubleshooting Table 12 Troubleshooting qPCR STR PCR results continued Observed problem Possible reason Suggested solution The sample IPC C is higher than the IPC C of the no template quantitation control NTC or of the quantitation standards for example if the sample IPC C is approximately two C greater than the NTC IPC C4 or the C of the standards or Weak amplification high C4 value and low AR value of the human and or male specific targets and no or weak amplification of the IPC The DNA concentration is above 25 ng uL Potential presence of PCR inhibitors If the DNA concentration is over 25 ng LL dilute the DNA extract then requantify the sample If the DNA concentrate is below 25 ng LL or if the diluted DNA eluate still produces high IPC C compared to the NTC or quantitation standards repurify the sample IMPORTANT Repurification may result in the loss of additional DNA Consider proceeding to amplification with a kit such as the AmpF STR MiniFiler PCR Amplification kit which is designed to obtain STR profiles from compromised samples such as those which may be inhibited and or degraded Before the next run To ensure correct pipetting clean and finger ti
21. EE HEN ewe eh eee 5 E Required instruments software and materials 00 0 eese 6 ErsiB h Procedures B For more information llis teen ene 7 Prepare qPCR Reagents and Labware Run Automated qPCR Setup Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution gPCR STR Setup System Getting Started Guide Chapter 1 The HID EVOlution gPCR STR Setup System HID EVOlution qPCR STR Setup System workflow HID EVOlution qPCR STR Setup System workflow The HID EVOlution gPCR STR Setup System automates qPCR and STR PCR reaction setup Table 1 below describes the steps performed by the user in a human identification HID workflow with the steps that involve the HID EVOlution qPCR STR Setup System highlighted in blue A detailed description of the steps is provided in Appendix C on page 89 Figure 1 on page 3 shows the integration of the HID EVOlution qPCR STR Setup System with the other instruments software and data files in the HID workflow Table 1 Overview of the automated HID workflow HID Workflow Covered in Chapter Extraction procedures are not included in this guide For automated extraction procedures see the PrepFiler Prepare Sample Extract DNA Automated Forensic DNA Extracti
22. Nw X P G7 oF o o OF 49 JS AP AS oF oF D OS dX dy KO QU SS T T T T Source DNA ng pL Figure 33 Precision Studies for the MiniFiler kit Figure 34 on page 120 shows representative STR profiles from a range of extracted DNA concentrations 10 uL of the 0 025 ng uL extracted DNA samples was transferred by the HID EVOlution system directly to the PCR reaction plate For extracted DNA samples with concentration gt 0 025 ng uL the concentration of each DNA sample was normalized to 0 1 ng uL and 10 uL of normalized sample was transferred by the HID HID EVOlution qPCR STR Setup System Getting Started Guide 119 E Appendix E Validation Experiments and Results Precision and reproducibility studies STR PCR amplification reaction setup scripts EVOlution system to the PCR reaction plate The x axis of plot indicates base pair size and the y axis indicates the RFU The source DNA concentration is as follows Panel A 50 ng uL Panel B 10 ng uL Panel C 5 ng uL Panel D 2 ng uL Panel E 1 ng uL Panel F 0 5 ng uL Panel G 0 1 ng uL and Panel H 0 025 ng uL Figure 34 SGM Plus kit electropherograms Figure 35 on page 121 and Figure 36 on page 121 show representative STR profiles from a range of extracted DNA concentrations 10 uL of the 0 025 ng uL extracted DNA samples was transferred by the HID EVOlution system directly to the PCR reaction plate For extracted DNA samples with concentration gt 0 025 ng uL
23. Quantifiler Human or Quantifiler Y Human Male kits Eight DNA standards in duplicate are placed in columns 1 and 2 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 4 Run Automated qPCR Setup 4 For more information 1 2 3 4 5 6 7 8 9 10 11 12 Figure 4 Plate layout for a combined setup using both Quantifiler Human and Quantifiler Y Human Male kits Eight DNA standards in duplicate are placed in columns 1 and 2 Human kit and in columns 7 and 8 Y Human Male kit For more information For information on HID EVOlution qPCR STR Setup System Getting Started Guide Running quantification scripts see the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 1 Running a Quantifiler Script How to import sample files see the Tecan HID EVOlution gPCR STR Setup System Application Manual Section 5 3 1 Running a Quantifiler Script Script error messages see the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 8 4 Application Software The EVOware software see the Tecan EVOware Standard EVOware Plus 2 1 Software Manual and Tecan EVOware Standard EVOware Plus 2 1 Software Getting Started Guide The qPCR Samples Report see the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 1 Running a Quantifiler Script and Sect
24. STR kit and plasticware that you are using Freedom EVOware software script selection for STR PCR Hue m ce a And the extracted DNA is in Samples run Use this script Identifiler kit reagents a 96 well plate 88 Identifiler plate esc 1 5 mL microcentrifuge tubes 88 Identifiler tubes esc Yfiler kit reagents a 96 well plate 87 Yfiler plate esc 1 5 mL microcentrifuge tubes 87 Yfiler tubes esc MiniFiler kit reagents 96 well plate 88 MiniFiler_plate esc 1 5 mL microcentrifuge tubes 88 MiniFiler_tubes esc COfiler kit reagents a 96 well plate 88 COfiler plate esc 1 5 mL microcentrifuge tubes 88 COfiler tubes esc Profiler Plus kit reagents a 96 well plate 88 Profiler plate esc 1 5 mL microcentrifuge tubes 88 Profiler tubes esc SEfiler Plus kit reagents a 96 well plate 88 SEfilerPlus plate esc 1 5 mL microcentrifuge tubes 88 SEfilerPlus tubes esc SGM Plus kit reagents a 96 well plate 88 SGMPlus plate esc 1 5 mL microcentrifuge tubes 88 SGMPlus tubes esc t This script is available in Freedom EVOware version 2 1 To upgrade scripts contact your local Tecan service organization See the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 10 Customer Support for contact information 2 Follow the directions in the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 2 Running a STR PCR Setup Script and the additiona
25. Setup Run STR PCR setup Global Req Amount ng Sets the target DNA amount to be added to each STR PCR reaction in the plate Note For samples below the target input amount the sample will not be processed unless you select the process checkbox for the individual sample or select Process All If you select this checkbox then 10 uL of extracted DNA will be added for a 25 uL STR reaction or 20 uL of extracted DNA will be added for a 50 uL STR reaction Qty range ng uL Sets the acceptable concentration range for extracted DNA samples Samples with concentrations above or below the range will not be included in the STR PCR reaction plate c Review the settings for individual samples in the sample list and edit the settings 1f necessary Note If you change any value s the system automatically performs the necessary recalculations Process Select or deselect an individual sample for processing Req Amt ng Sets the target DNA amount to be added to the STR PCR reaction for a specific extracted DNA sample Optional but recommended When you are done reviewing and editing the information in the Sample Normalization Adjustment window take a screenshot to capture the information then name and save the screenshot IMPORTANT You can use this information later to determine the dilution ratio used for each sample the amount of extracted DNA removed from the extracted DNA plate or tubes and the ta
26. Software Runtime Controller Manual PN 394329 Note For additional documentation see How to obtain support on page x HID EVOlution qPCR STR Setup System Getting Started Guide 149 Documentation Obtaining information from the Help system Obtaining information from the Help system The Tecan Freedom EVOware v2 1 has a Help system that describes how to use each feature of the user interface Access the Help system by doing one of the following Click in the toolbar of the Tecan Freedom EVOware 2 1 window Select Help gt Contents and Index Press F1 You can use the Help system to find topics of interest by Reviewing the table of contents Searching for a specific topic Searching an alphabetized index Note The Help system is specifically for the Tecan Freedom EVOware 2 1 system not the HID EVOlution gPCR STR Setup System Changing the Freedom EVOware 2 1 system parameters could lead to problems running qPCR STR PCR scripts Consult with a system administrator before making any system changes Send us your comments 150 Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is for submitting comments and suggestions relating only to documentation To order documents download PDF files or for help with a technical question see How t
27. System and software refer to the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide 3 Import the 7500 Setup file into the Applied Biosystems 7500 SDS Software plate document for use during the quantitation run as follows a In the SDS software select File New to create a new Standard Curve Absolute Quantitation plate document then in the new document wizard select Finish b With a blank plate document open select File Import Sample Set Up c Browse to locate the 7500 Setup file select the file ReactionPlatel txt or lt barcode gt txt then click Open d Modify the instrument protocol Delete the Stage 1 hold step 50 C for 2 minutes and change the Sample Volume to 25 HID EVOlution qPCR STR Setup System Getting Started Guide 41 H Chapter 5 Perform qPCR and Review Results Perform qPCR 4 Save the plate document a Select File gt Save As b In the Save as dialog box Enter a file name in the File name field Do not add an extension to the file name Select SDS Document sds from the Save as type drop down list Click Save Perform qPCR Perform qPCR as described in the Applied Biosystems Quantifiler Kits User s Manual Analyze and export the qPCR results When the qPCR run is complete use the following procedures to Analyze and review the qPCR results Export the 7500 Results file 1 Analyze and review the 7500 results a I
28. X 5 n poc D c EC SS SS SS SS SS SS DNA Quantity ng ul R S QY S e 4 Figure 26 Precision study for automated Quantifiler Human and Y Human Male kit reaction setup using extracted DNA from a 96 well plate 112 HID EVOlution qPCR STR Setup System Getting Started Guide Table 19 Average concentration and standard deviation for automated reaction setup using the Quantifiler Human and Y Human Male DNA Quantification Kits and each of the following scripts Results In determining the accuracy of the Quantifiler Human and Y Human Male Appendix E Validation Experiments and Results Precision and reproducibility studies qPCR reaction setup scripts Quantification assay and reaction setup using the combined qPCR setup script on the HID EVOlution system the average concentrations and standard deviations were calculated see Table 19 QuantifilerHuman plate esc QuantifilerHuman tubes esc QuantifilerY plate esc QuantifilerY tubes esc QuantifilerHumanY plate esc and QuantifilerHumanY tubes esc Human Human Y plate Y tube pen Combo y Hd Combo plate tube Human plate Y Human tube Y N N 36 N 36 N 12 N 12 N 5 N 5 N 5 N 5 Average concentration ng pL 0 025 ng uL 0 037 0 035 0 024 0 038 0 029 0 033 0 025 0 028 0 125 ng uL 0 054 0 067 0 062 0 097 0 056 0 080 0 057 0 074 0 1 ng uL 0 100 0 127 0 115 0 161 0 092 0 144 0 113 0 167 1 ng uL 0 977 1 125 1 14
29. are done with amplification thermal cycling prepare for CE 66 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis About the CE Setup file About the CE Setup file The HID EVOlution system software generates a plate file CE Setup file for each run The file contains sample information that you can import to the Data Collection Software plate document for the STR run on the CE instrument 3130 3130x Genetic Analyzer computer By default the HID EVOlution qPCR STR Setup System names the CE Setup file C HIDEVOlution_qPCRSTRfiles AB3 130Input STRplate_ lt rundate gt _ lt runtime gt txt or C HIDEVOlution_qPCRSTRfiles AB3130Input lt barcode gt _ lt rundate gt _ lt runtime gt txt The HID EVOlution system automatically archives the CE Setup file in the C HIDEVOlution_qPCRSTRfiles AB3130Input Archive folder when you start the next qPCR or STR PCR setup run If necessary create shortcuts on your desktop to the C HIDEVOlution_qPCRSTRfiles AB3130Input and C HIDEVOlution_qPCRSTRfiles AB3130Input Archive folders p f sTRplate 20070717 163314 txt Notepad File Edit Format View Help r ontainer Name Description STRplate 20070717 163314 AAppServer AppInstance GenemMapper well Sample Name IDl IDL ID2 ID2 ID3 ID3 ID4 ID4 ID5 ID5 ID6 ID6 ID7 ID7 ID8 ID8 ID9 ID9 ID10 ID10 ID11 ID11 ID12 ID12 Positive Negative ladder
30. averages Precision studies for Yfiler kit peak height averages e Precision studies for MiniFiler kit peak height averages SGM Plus kit STR profiles Profiler Plus kit STR profiles COfiler kit STR profiles Figure 29 below and Figures 30 and 31 on page 118 show average peak height across different starting concentrations Positive refers to the positive control used in the experiment Red bars represent sample data generated when processing DNA extracts from a 96 well plate source vessel and green bars represent sample data generated when processing DNA extracts from tubes source vessels Profiler Plus ID Plate and Tube ee Bl Plate Bl Tube 2500 J S 2000 9 1500 3 Ea algal m H 1000 Lh B J 3 H H i E o PT 7 1g 500 4 0 025 01 05 1 2 5 10 50 Positive Source DNA ng uL Figure 29 Precision studies for the Profiler Plus kit HID EVOlution qPCR STR Setup System Getting Started Guide 117 E Appendix E Validation Experiments and Results Precision and reproducibility studies STR PCR amplification reaction setup scripts COfiler Plate and Tube 3000 Bil Plate B Tube 2500 4 w tz m S 2000 1500 p a z o T S 1000 H T a 504 dm 0 T T T T T T T T T 0005 01 05 1 2 5 10 50 Positive Source DNA ng uL Figure 30 Precision studies for the COfiler kit SEfiler Plate and Tube
31. d ces m B Run STR PCR amplification 0 0 cece ae 66 B About the CE Setup Mle jos554 eben dx bea nce riot kacileg d 67 E Transfer and import the CE Setup file 0 2 0 0 eee eee eee 68 B Prepare the CE Plates sius e RR xe e EUER OPE WEE RS 68 Pre Run Procedures E For more information 4 his enh bie ede eh eee PREC A COE PER E EE VER ERN 69 Prepare qPCR Reagents and Labware Run Automated qPCR Setup Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution gPCR STR Setup System Getting Started Guide 65 Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis Run STR PCR amplification Run STR PCR amplification Refer to the appropriate AmpF STR kit documentation for amplification procedures Document Part number AmpF STR COfiler PCR Amplification Kit User s Manual 4306116 AmpF STR Identifiler PCR Amplification Kit User s Manual 4323291 AmpF STR MiniFiler PCR Amplification Kit User s Manual 4374618 AmpF STR Profiler Plus PCR Amplification Kit User s Manual 4303501 AmpF STR SEfiler Plus PCR Amplification Kit User s Manual 4385739 AmpF STR SGM Plus PCR Amplification Kit User s Manual 4309589 AmpF STR Yfiler PCR Amplification Kit User s Manual 4358101 When you
32. enter or information import the sample name and information for each extracted DNA sample The sample information is used by the HID EVOlution qPCR STR Setup System to Set up the reaction plate Generate a Samples Report at the end of the run The report records each extracted DNA sample starting position in a plate or in tubes and each sample final position in the reaction plate Generate a text file containing the sample information qPCR reaction setup run Generates a 7500 Setup file that you can import into the SDS software plate document to define the parameters of the 7500 Real Time PCR System run STR PCR reaction setup run Generates a CE Setup file that you can import into the Data Collection Software to define the parameters of the 3130 3130x Genetic Analyzer run Options for entering You have several options for entering sample information You can sample information P Automatically capture sample information by having the system scan barcodes on the plates and or tubes Select barcodes compatible with the PosID 3 then make sure the barcodes are correctly placed when you set up the worktable See the Tecan HID EVOlution M qPCR STR Setup System Application Manual Section 4 6 Barcodes for details For Barcodes on 96 well plates After the plate barcode is scanned you must manually enter or import the sample name and information for each well in the plate See the
33. extracted DNA SAIN Esse PME Cx 96 How the HID EVOlution system dilutes asample 0 00005 96 Order of processing and placement of D1 and D2 in the pre dilution plates 98 Dilution protocols with examples 0 00 ccc cee eens 98 About the HID EVOlution gPCR STR Setup System dilution protocols 98 How to determine the volume of extracted DNA sample used in each reaction 98 Dilution protocol examples 12 0 0 0 0 ccc eee eens 99 Dilution protocol tables 20 0 0 cece cece eens 99 HID EVOlution gPCR STR Setup System Getting Started Guide 93 Appendix D Dilution Protocols DNA input amount must be normalized before STR PCR amplification DNA input amount must be normalized before STR PCR amplification If the concentration ng uL of an extracted DNA sample is higher than the target DNA input ng for the AmpF STR PCR Amplification kit then the extracted DNA sample must be diluted to normalize the DNA input in the STR PCR reaction plate The HID EVOlution system automates DNA input normalization using a one or two step dilution process The workflow and examples of the dilution ratios used to achieve this are in the sections that follow Terms used in the Freedom EVOware software Required amount of DNA Quantity Configured volume The target amount of DNA ng you want in each reaction in the STR PCR reaction plate The concentration of the extracted DNA ng uL as shown in
34. precision validation 126 HID EVOlution gPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Complete system check and precision study oo Sample qPCR setup on the HID EVOlution System qPCR Samples Report output ile input 7500 Setup file output ee 7500 Setup file gt Perform real time qPCR on the 7500 instrument input M Perform data analysis using the SDS software 7500 Results file J output 7500 Results file input Dilution and normalization of sample concentrations gt STR Samples Report output STR PCR amplification reaction setup CE Setup file output qPCR STR Sample on the HID EVOlution System file input r Perform STR PCR on the 9700 thermal cycler i CE Setup file gt Perform CE on the 3130x Genetic Analyzer input 4 Perform data analysis using the GeneMapper Software LE STR results Figure 39 HID EVOlution system process HID EVOlution gPCR STR Setup System Getting Started Guide 127 E Appendix E Validation Experiments and Results Complete system check and precision study Results The quantification results obtained Figure 40 were similar to the expected results across a range of concentrations from 50 ng uL to 25 pg uL Quantification results were as expected for the sample DNA concentrations Sam
35. sample prepared with the Identifiler kit for processing the HID EVOlution system transfers 10 uL of the sample containing 0 25 ng of DNA to the STR PCR plate Example 1 step dilution Using Table 15 on page 100 if you have a sample concentration of 1 2 ng uL and you are targeting a total input amount of 1 ng the sample will need to be diluted 12 times The closest dilution protocol to this is 1 13 3 and so the sample will be diluted 1 13 3 times resulting in a final concentration of 0 902 ng uL To perform this dilution the HID EVOlution system transfers 3 uL of sample from the original DNA sample plate and adds it to 37 uL of TE in the predilution plate D1 The system will then thoroughly mix the TE and sample DNA before transferring 10 uL from D1 to the STR PCR plate D1 to PCR A single dilution step is sufficient for up to a 20 times dilution for a 10 uL addition Table 15 on page 100 or up to a 40 times dilution for a 20 uL addition Table 16 on page 101 Example 2 step dilution A more concentrated sample needs stepwise dilutions to economize sample usage but still get a thoroughly mixed dilution For example if you have a sample containing 3 9 ng uL it will need to be diluted 36 times to be normalized for a 10 uL addition to the STR PCR plate To perform this stepwise dilution the HID EVOlution system starts by transferring 2 uL of the original DNA sample and mixes it with 38 uL of TE in the first dilution D1 Th
36. sample requires rather the two plates provide enough wells in the event that all samples require a two step dilution The D2 dilutions do not necessarily occur in a second plate They can occur in the first plate in wells that are adjacent to the D1 dilutions Additionally when the samples that require a two step dilution are being normalized the HID EVOlution system processes them starting in a new column of the plate This could leave some wells empty following the normalization of one step diluted samples Dilution protocols with examples 98 About the HID EVOlution qPCR STR Setup System dilution protocols How to determine the volume of extracted DNA sample used in each reaction In order to cover a dynamic range for dilutions from 1 1 to 1 4000 or 1 8000 for the kits that use 20 uL of sample different volumes are used in the dilution steps The dilution protocols used by the HID EVOlution qPCR STR Setup System were developed based on the following considerations Minimized DNA extract consumption pipetting accuracy and mechanical characteristics of the pipetting system The dilution volumes are based on the physical properties of the system increments of volumes in 83 3 nL steps while minimizing sample consumption and maximizing pipetting accuracy The maximal deviation from calculated to executed dilution is less than 15 at the end of each dilution range The amount of the original extracted DNA sa
37. samples that are outside the acceptable quantity range Only those samples with data that successfully merged with the 7500 Results file and which meet the conditions for normalization are automatically selected for dilution and STR reaction setup You can manually select or deselect samples for processing and edit the acceptable quantity range and the target DNA input amount The sample dilution calculations in the Sample Normalization Adjustment window are based on the STR PCR amplification reaction volume the sample quantification data and the input amount of DNA that you want to target Req Amt ng The software selects the appropriate dilution protocol that brings the DNA input amount after dilution closest to the required DNA target amount After dilution 10 uL of diluted DNA for 25 uL reactions or 20 uL of diluted DNA for 50 uL reactions is added to each PCR reaction b Review the global settings for all samples in the sample list and edit the settings if necessary Note If you change any value s the system automatically performs the necessary recalculations Process All Select this checkbox to process all samples for STR PCR amplification setup To return to the original settings select Cancel then select View to re open the window De select the Process AII checkbox to deselect a samples for processing HID EVOlution qPCR STR Setup System Getting Started Guide 59 60 Chapter 7 Run Automated STR PCR
38. scripts Precision and reproducibility studies qPCR reaction setup scripts Quantifiler Human reaction setup precision and reproducibility study Experiment Automated setup of Quantifiler Human kit reactions from a plate 108 qPCR reaction setup precision and reproducibility studies were performed with a single female genomic DNA sample The genomic DNA was quantified using the Quantifiler Human DNA Quantification kit and based on the results the original female genomic DNA stock was manually diluted to 0 025 0 05 0 1 1 0 5 0 and 25 0 ng uL of DNA Each concentration was then transferred either into microcentrifuge tubes or into a 96 well plate that served as a source DNA vessel The Quantifiler kit reagents twelve replicates of each sample DNA concentration and the DNA standard dilution series were dispensed by the HID EVOlution system into a qPCR reaction plate qPCR was run on a single 7500 Real Time PCR System Each study plates or tubes was repeated three times Samples containing 0 025 0 05 0 1 1 0 5 0 and 25 0 ng uL of DNA in a 96 well source plate were quantified for 12 replicates in three runs In Figure 22 the red boxes represent plate study 1 P1 the green boxes represent plate study 2 P2 and the blue boxes represent plate study 3 P3 Quantifiler Human Precision and Reproducibility Study 3 9 F 1 ng ul a F e D 1ng ul 0 01ng ul Plate P1P2 P3 P1 P2 P3 Pi P2 P
39. setups One half tray of 200 uL DiTis for 96 reactions One and one half trays of 50 uL DiTis for 96 reactions e HID EVOlution gPCR STR Setup System specific carriers Two 96 well metal microplate plate adapters Up to five 16 position tube carriers with vertical cap storage if extracted DNA samples are in tubes Optional Barcodes for DNA sample plate or tubes and qPCR reaction plate Make a list of the lot numbers and expiration dates of the Quantifiler kit components that will be used in the run for entry into the HID EVOlution qPCR STR Setup System software Set up the carriers and racks according to the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 4 3 3 Set Up Carriers and Racks Oo Start up the system and perform routine maintenance including running the appropriate maintenance scripts according to the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 4 3 2 Prepare the Instrument section 5 1 Starting the System and Before each run Run maintenance scripts on page 12 in this guide Determine the amount of reagents and place the reagents and DNA standard dilution series if previously prepared in the reagent block as described in Set up reagents on page 25 Place the reagents labware and samples on the worktable as described in Set up the labware on the worktable on page 30 24 HID EV
40. steps performed by the user and by the HID EVOlution gPCR STR Setup System are described in the table and text below Table 13 Overview of the automated HID workflow HID Workflow Covered in Chapter Extraction procedures are not included in this guide For automated extraction procedures see the PrepFiler Prepare Sample Extract DNA Automated Forensic DNA Extraction Kit Getting Started V Guide Perform Quantitative PCR 1 Perform routine maintenance 2 Pre Run Procedures on page 9 2 Prepare samples qPCR reagents and system 3 Prepare qPCR Reagents and Labware on page 23 3 Run automated qPCR reaction setup 4 Run Automated qPCR Setup on page 33 4 Import a SDS plate record 5 Perform qPCR and Review Results on page 39 5 Run qPCR and review results 5 Perform qPCR and Review Results on page 39 Perform STR PCR Amplification 1 Perform routine maintenance 2 Pre Run Procedures on page 9 2 Prepare samples STR PCR reagents and system 6 Prepare STR PCR Reagents and Labware on page 45 3 Run automated DNA normalization and STR PCR 7 Run Automated STR PCR Setup on page 57 reaction setup 4 Run STR PCR amplification 8 Perform STR PCR and Set Up Capillary Electrophoresis on page 65 Perform Capillary Electrophoresis Genetic Analysis 1 Import a CE plate record 8 Perform STR PCR and Set Up Capillary Electrophoresis on page 65 2 Set up CE reactions P pu
41. xe cued Shean c acus mie ae te ses 71 Reagent Block Configurations sssaaa aaaeeeaa 75 Quantifiler DNA Quantification Kits eon rod iml thet kk ede ohn wie eB 76 Quantifiler Human Kit with pre prepared standards 0 00 eee eeeeee 76 Quantifiler Human Kit with system prepared standards 000e0 eens 77 Quantifiler Y Human Male Kit with pre prepared standards 78 Quantifiler Y Human Male Kit with system prepared standards s 79 Quantifiler Human and Y Human Male Kits with pre prepared standards 80 Quantifiler Human and Y Human Male Kits with system prepared standards 81 AmpF STR PCR Amplification Kits 00 c cece hh m e 82 AmpF STR COfiler PCR Amplification Kit 2 0 0 0 cc ccc eee eee 82 AmpFiSTR Identifiler PCR Amplification Kit 2 0 0 0 00 00 c cece eee eee 83 AmpF STR MiniFiler PCR Amplification Kit 0 000 c cee e 84 HID EVOlution gPCR STR Setup System Getting Started Guide Appendix C Appendix D Appendix E Appendix F HID EVOlution gPCR STR Setup System Getting Started Guide Contents AmpF STR Profiler Plus PCR Amplification Kit 0 0 0 ccc eee eee ee 85 AmpF STR SEfiler Plus PCR Amplification Kit llle 86 AmpF STR SGM Plus PCR Amplification Kit 2 0 0 0 0 cece eee eee ee 87 AmpF STR Yfiler PCR Amplification Kit 0 0 0 0 ccc cece cece ene 88 HID EVOluti
42. 10 5 uL e Quantifiler PCR Reaction Mix 12 5 uL Note Include excess reactions in the calculations to compensate for the loss that occurs during reagent transfers HID EVOlution qPCR STR Setup System Getting Started Guide 135 Appendix F Automation Guidelines qPCR reaction setup automation guidelines Upon completing 136 the automated process To prepare the reactions 1 Dispense the required volume of primer mix into a separate empty tube for Master Mix preparation Dispense the required volume of PCR reaction mix into the Master Mix tube then mix well Dispense 23 uL of Master Mix into each reaction well Add 2 uL of DNA standard or sample to each well IMPORTANT As you add standards or samples be sure to mix the components of the reaction thoroughly Applied Biosystems recommends running duplicates of the eight DNA quantification standards for each assay and on each reaction plate For plate setup examples see About the qPCR reaction plate layout on page 36 When you are done with the automated process Seal the reaction plate with the clear adhesive film Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles Run the PCR reaction as described in the Quantifiler Kits User s Manual Remove all reagents from worktable Close the sample tubes to prevent contamination and evaporation Flush the liquid system Clean the work
43. 2 1 5 mL tube containing Tj Ep buffer 3 Empty VWR tube for master mix preparation 4a 4b AmpF STR PCR Reaction Mix 5 SGM Plus Primer Mix 6 AmpliTaq Gold DNA polymerase t You must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors HID EVOlution qPCR STR Setup System Getting Started Guide 87 Appendix B Reagent Block Configurations AmpF amp STR PCR Amplification Kits AmpF STR Yfiler PCR Amplification Kit e 4p 6 Figure 20 Reagent block configuration for Yfiler kit Legend 1a Control DNA 007 diluted as necessary See step 4 on page 52 1b Control DNA 9947A diluted as necessary See step 4 on page 52 2 1 5 mL tube containing T 5E buffer 3 Empty VWR tube for master mix preparation 4a 4b Yfiler PCR Reaction Mix 5 Yfiler Primer Mix 6 AmpliTag Gold DNA polymerase t You must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors 88 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix C HID EVOlution qPCR STR Setup System Detailed Workflow Description The
44. 2 HID EVOlution qPCR STR Setup System Getting Started Guide The HID EVOlution qPCR STR Setup System Pre Run Procedures Chapter 3 Prepare qPCR Reagents and Labware Run Automated qPCR Setup Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 3 Prepare qPCR Reagents and Labware This chapter provides procedures to prepare reagents labware and the Tecan Freedom EVO workstation for quantitative PCR qPCR reaction setup B Review pre run checklist for qPCR reaction setup 0 00 e ee eee 24 M Set up reagents 2 0 ec Rh eens 25 About minimum required reagent volumes 02 0 00 eee eee eee 25 Determine required reagent volumes 00 000 c cece cece eee nee 26 Place reagents in the qPCR reagent block 00 00 eee eee ee 29 BW Set up the labware on the worktable 0 0 0 eee 30 23 Chapter 3 Prepare qPCR Reagents and Labware Review pre run checklist for GPCR reaction setup Review pre run checklist for qPCR reaction setup Cool the qPCR reagent block to 4 C before use to help keep reagents cool on the worktable It is recommended when not in use that you store reagent blocks in a refrigerator at 4 C O Optional Create a qPCR STR Sample file that you
45. 2 on page 32 as a guide CAUTION For important safety information related to the use of the Tecan Freedom EVO instrument refer to the manufacturer s instrument documentation 1 Set up the DiTis as described in the Tecan HID EVOlution GPCR STR Setup System Application Manual Section 4 3 5 Set Up Plasticware and Samples on the Workstation IMPORTANT Ifthe DiTi trays are not correctly set up in the two carriers for example if the 50 uL DiTis are placed in the 200 uL DiTis position the LiHa may crash or pipetting errors may result If there are no DiTis where the software expects them the run will pause until you replenish the DiTis During this pause the samples may become unstable To prevent this from occurring use full DiTi trays and run the script Set DiTi Position 200 or Set DiTi Position 50 to set all DiTis to position 1 Confirm that you have correctly loaded the reagent block see Place reagents in the qPCR reagent block on page 29 remove the caps from the prepared reagents then place the loaded reagent block on grid 15 site position 1 Ifthe HID EVOlution system is preparing the DNA standards place 6 mL Human or Y Human Male or 7 mL Human and Y Human Male of T Eo buffer into a 100 mL trough see Figure 2 item 7 then place the trough on grid 14 site position 2 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 3 Prepare qPCR Reagents and Labwa
46. 25 pL reaction volume Required Extracted Diluted Required Required m Available Volus B DNA Standard Excess Dead Minimum Required Volume for Reagent Volume in Raaton Samples Samples Reactions Volume 80 extracted DNA samples and 9 Full Tube of per Run per Run per Run per Tube 16 diluted standards Reagent Ax B C D E A B Cc D E Quantifiler Human or Y 1 4 mL 11 55 uL up to 80 16 3 50 uL 1194 uL in one tube Human Male Primer Mix Quantifiler PCR 5mL 13 75 uL up to 80 16 3 50 uL 1412 uL in one tube Reaction Mix A 50 uL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips 11 55 ul reaction x 80 16 3 reactions 50 uL tube 1193 45 uL in one tube If you divide the minimum volume of Primer Mix across multiple tubes each tube must contain a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For example place at least 1000 uL in the first tube and 240 pL in the second tube 13 75 ul reaction x 80 16 3 reactions 50 uL tube 1411 25 uL in one tube Table 7 Reagent volumes for a combined qPCR reaction setup using both the Quantifiler Human and Y Human Male Quantification Kits in one qPCR reaction plate 25 pL reaction volume Banuir d Extracted Diluted Required R
47. 3 1 467 0 933 1 154 1 156 1 554 5 ng uL 4 911 5 765 5 003 6 433 4 990 5 314 6 116 7 038 25 ng uL 26 314 31 417 23 637 28 493 28 608 25 590 36 960 35 120 Standard deviation 0 025 ng uL 0 02 0 01 0 009 0 014 0 008 0 007 0 010 0 018 0 125 ng uL 0 01 0 01 0 009 0 024 0 003 0 013 0 005 0 025 0 1 ng uL 0 02 0 02 0 025 0 019 0 013 0 025 0 015 0 027 1 ng uL 0 09 0 06 0 050 0 080 0 040 0 042 0 042 0 141 5 ng uL 0 25 0 34 0 198 0 242 0 217 0 068 0 269 0 208 25 ng uL 2 31 2 15 0 751 0 800 0 521 0 621 1 113 1 423 HID EVOlution qPCR STR Setup System Getting Started Guide 113 Appendix E Validation Experiments and Results Precision and reproducibility studies STR PCR amplification reaction setup scripts Precision and reproducibility studies STR PCR amplification reaction setup scripts Identifiler and SGM Plus STR PCR amplification reaction setup precision and reproducibility studies 114 Experiment Results Precision and reproducibility studies were conducted to evaluate the sample dilution and liquid transfer steps of the automated STR PCR amplification setup protocols Each AmpF STR kit script was verified for functionality precision and reproducibility This study was performed using 0 025 0 1 0 5 1 2 5 10 and 50 ng uL DNA samples that were prepared on the HID EVOlution system and quantified using the Quantifiler Human kit on the 7500 Real Time PCR System Eleven replicates of each DNA concen
48. 3 P1P2P3 P1 P2 P3 PI P2 PS DNA Source ng ul 0 025 0 05 0 1 10 5 0 25 0 Figure 22 Precision and reproducibility study for the automated reaction setup of Quantifiler Human kit reactions using extracted DNA from a 96 well plate HID EVOlution qPCR STR Setup System Getting Started Guide Automated setup of Quantifiler Human kit reactions from tubes Quantifiler Human kit results eo Average DNA Quantity ng ul HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results Precision and reproducibility studies qPCR reaction setup scripts Samples containing 0 025 0 05 0 1 1 0 5 0 and 25 0 ng uL of DNA in 1 5 mL microcentrifuge tubes were quantified for 12 replicates in three runs In Figure 23 the red boxes represent tube study 1 Tubel the green boxes represent tube study 2 Tube2 and the blue boxes represent tube study 3 Tube3 Quantifileri Human Precision and Reproducibility Study 100 Container zi 10 c gt Dni S 1 3 E lt 2 a 0 1 0 01 Tube KYAV AD KYKVK RVAV RD RV RVRD RVAV AD RY RV RY DNA Source ng ul Y S S S ng ul 4 ge ey pu ey Ls Figure 23 Precision and reproducibility study for the automated setup of Quantifiler Human kit reactions using extracted DNA from microcentrifuge tubes Figure 24 below and Table 18 on page 110 show the average DNA concentration across a range of DNA concentrations and c
49. 60 1 5 148 5 117 76 2 24 20 600 00 738 46 1 6900 1 5 148 5 118 26 1 74 20 738 46 800 00 1 8000 1 5 148 5 118 5 1 50 20 102 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results This appendix covers pau C 104 Materials and methods 00 cece cee eee 105 Precision and reproducibility studies DNA standard dilution series preparation and reaction Setups s osse nee Rand adn vo ea ARR e XU aye 107 Precision and reproducibility studies qPCR reaction setup scripts 108 Quantifiler Human reaction setup precision and reproducibility study 108 Quantifiler Y Human Male kit precision study 0 0005 110 Combined Quantifiler Human and Quantifiler Y Human Male reaction setup precision study cess ctisete tetant ee eeke eae n 111 Precision and reproducibility studies STR PCR amplification reaction setup SCEIDIS ia aeaa oe E aA E a a E E Aa d RH 114 Identifiler and SGM Plus STR PCR amplification reaction setup precision and reproducibility studies 0 0 lisse ees 114 Other AmpF STR kits Supplemental precision and reproducibility studies 117 Identifiler and SGM Plus STR PCR amplification reaction setup accuracy qun MMC CIT 122 Complete system check and precision study 00 00 e eee euee 126 Concordance and position ID confirmation study 0 05 131 Contamination study
50. 68 2 88 3 83 36 17 10 54 00 72 00 1 630 2 88 2 85 37 15 10 72 00 93 91 1 837 2 88 2 15 37 85 10 93 91 120 00 1 1080 2 88 2 50 57 50 10 120 00 154 29 1 1337 2 88 2 02 57 98 10 154 29 205 71 1 1800 2 88 2 00 78 00 10 205 71 266 67 1 2480 1 5 148 5 2 42 57 58 10 266 67 342 86 1 2990 1 5 148 5 2 01 57 99 10 342 86 400 00 1 4000 1 5 148 5 2 00 78 10 100 HID EVOlution gPCR STR Setup System Getting Started Guide Appendix D Dilution Protocols Dilution protocols with examples Table 16 Dilution protocols for 20 pL transfer Columns 1 and 2 apply only for a DNA target input amount of 1 ng If your sample concentration is First Dilution Mixture D1 Second Dilution Mixture D2 Volume between Then the D1 or D2 dilution added to ratio is Volume or Volume from SIR PC R Min Max extracted TE added to D1 added to TE added to reaction ng uL ng uL DNA added D1 pL D2 uL D2 uL plate pL to D1 pL 0 000 0 115 1 1 Sample is transferred directly from the extracted DNA plate or 20 tube to the STR PCR plate 0 115 0 155 143 3 19 77 6 89 0 0 20 0 155 0 209 1 1 8 18 34 15 0 0 20 0 209 0 281 1 2 4 16 34 23 66 0 0 20 0 281 0 378 1 3 3 12 16 27 84 0 0 20 0 378 0 511 1 4 4 9 31 0 0 20 0 511 0 686 1 6 6 66 33 34 0 0 20 0 686 0 906 1 8 5 35 0 0 20 0 906 1 171 1 10 4 3 84 36 16 0 0 20 1 171 1 500 1 13 3 3 37 0 0 20 1 500
51. 7 Chapter 8 Appendix A Appendix B Perform qPCR and Review Results ls 39 About the 7500 Setup file 2l 40 Transfer and import the 7500 Setup file 000 cee ees 41 Perform GPCR uoa esee RR Pe eho gon Cu RU ee ie dr um ER E RU RS 42 Analyze and export the qPCR results 0 00 42 For more information 00 000 hh hn 43 Prepare STR PCR Reagents and Labware 45 Review pre run checklist for STR PCR reaction setup 0 00 00 eee eee 46 Determine reagent volumes lslleseeeeeeeeel el meh 47 Set UP reagents cs ELE EEUU eS LV MER 52 Set up the labware on the worktable 0000 cee sees 54 Run Automated STR PCR Setup 0 00 cee eee eee 57 R n STR POR Set p uon exce rts tue ee Re eese eared Ramen O Bt E 58 Perform post run tasks as em bed N RR te Se ede ARE UE Rs 61 About the STR PCR plate layout esseeeeeeeee re 62 For more informatlon z sis axo eR E S eee tate exe Re ge Paneer T 63 Perform STR PCR and Set Up Capillary Electrophoresis 65 Run STR PCR amplification llllllllleelel II 66 About the GE Setup flle sco Gisele RE REIHE ek eee SE EW eee Busen eee 67 Transfer and import the CE Setup file 2er 68 Prepare the CE Plate sn 2p e ERE ates bee E RI ERG NU ERU PEE ONDRA 68 For more information ieee ia caiat ia da di e E hh hn 69 TIOBDISSHOOBFPD s eces dcm ac eae iag
52. 90 pL in the first tube and 1000 pL in the second tube ttiYou must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors Table 11 COfiler Profiler Plus SGM Plus kits reagent volumes 50 pL reaction volume for STR PCR amplification ie 5 Required Extracted Control Required Required E Available Volume DNA Samples Excess Dead o Reagent Volume in per Samples er ES Reactions Volume a E 9 Full Tube Reactiont per Run p per Run per Tube Minimum Required Volume for E of Reagent 88 Samples and 2 Controls d A B Cc D E AmpF STR Primer Set 1 1 mL 12 1 uL up to 88 2 0 4 50 uL A x B C D E e x 1144 uL ro 2 AmpF STR PCR Reaction Mix 1 1 mL 23 1 uL up to 88 2 0 4 50 uL A x B C D E EA 2139 uLSS in one tube lt AmpliTaq Gold DNA Polymerase 50 uL 100 uL combined in one tube for 1 to 88 samples plus 2 controls 100 uL combined in one tubet t 3 O AmpF STR Control DNA 9947A 300 uL 70 uL for 1 to 88 samples plus 2 controls 70 uL a or 007 T40Eo 1 buffer in reagent block NA 70 uL for 1 to 88 samples plus 2 controls 70 uL S T4o9Eo 4 buffer in trough NA 30 mL in trough for 1 to 88 samples plus 2 controls 30 mL in trough amp Q oO eremqe pue sjueBeeu Hod HIS aedaly 9 Je1deuo seuinjoA u be eu
53. Automation Guidelines STR PCR reaction setup automation guidelines 4 Dispense the required volume of Master Mix into each reaction well See the table below Master Mix for reaction wells i o eee VM Profiler SEfiler SGM S COfiler Identifiler MiniFiler Plus Plus Plus Yfiler Master Mix 30 0 15 0 15 0 30 0 15 0 30 0 15 0 5 Dispense the required volume of DNA sample into each reaction mix then mix well See the table below DNA sample volume for reaction wells x Ti Profiler Identifiler MiniFiler Plus COfiler PI TM PI Yfiler DNA sample volume minimum 20 0 10 0 10 0 20 0 10 0 20 0 10 0 Upon completing the automated process 6 Add DNA controls PTC and NTC as specified in the particular kit in use Note Remember that the Yfiler kit uses an extra PTC When you are done with the automated process Seal the reaction plate with clear adhesive film Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles e Run the PCR reaction as described in the kit specific User s Manual Remove all reagents from worktable Close the sample tubes to prevent contamination and evaporation Flush the liquid system Clean the worktable and carriers to avoid cross contamination HID EVOlution qPCR STR Setup System Getting Started Guide 139 F Appendix F Automation Gui
54. DNA in tubes 15 worktable layout 56 worktable setup 54 SWGDAM validation experiments and results 103 system configuration 4 T training information on x troubleshooting CEsignal 72 73 entering or importing sample name and information 59 high sample IPC Cy 72 low DNA yield 71 tubes for extracted DNA samples 15 V validation experiments and results 103 W WARNING description ix waste disposal guidelines 144 waste profiles description 144 workflow detailed 89 overview vii 2 worktable layout qPCR reaction setup 30 STR PCR reaction setup 56 Y Yfiler kit reagent block configuration 88 required reagent volumes 49 154 HID EVOlution qPCR STR Setup System Getting Started Guide Part Number 4426903 Rev B 08 2010 International Sales Applied Headquarters Bi t 5791 Van Allen Way Carlsbad CA 92008 USA For our office locations please call the division losys ems Phone 650 638 5800 Toll Free 800 345 5224 headquarters or refer to our Web site at www appliedbiosystems com www appliedbiosystems com about offices cfm
55. Getting Started Guide P plate layout qPCR reaction plate 36 STR PCR reaction plate 62 plate setup files export 7500 Results file 42 import 7500 Setup file 41 import CE Setup file 68 preparing AmpliTaq Gold DNA Polymerase 52 control DNA 52 PrepFiler Automated Forensic DNA Extraction Kit 2 89 Profiler Plus kit reagent block configuration 85 required reagent volumes 51 Q qPCR 7500 Results file 42 reaction plate layout 36 qPCR reaction setup about reagent volumes 25 maximum samples per run 13 required materials 24 run a script 34 set up extracted DNA in a 96 well plate 14 set up extracted DNA in tubes 15 set up reagent block 29 worktable layout 30 qPCR STR Sample file create 18 in HID workflow 3 Qty in Sample Normalization Adjustment window 94 Quantifiler DNA Quantification Kits reagent block configuration 76 77 78 79 80 81 reagent volumes 26 28 scripts 34 supported for use with HID EVOlution qPCR STR Setup System 5 R radioactive waste handling 145 reaction plate layout qPCR 36 STR PCR 62 reagent block configuration COfiler kit 82 Identifiler kit 83 MiniFiler kit 84 Profiler Plus kit 85 HID EVOlution gPCR STR Setup System Getting Started Guide Index reagent block configuration continued Quantifiler Human and Quantifiler Y Human Male kits combined setup 80 81 Quantifiler Human kit 76 77 Quantifiler Y Human Male kit 78 79 SEfiler Plus kit 86 SGM Plus
56. ID EVOlution driver v1 0 0 12 and the Tecan Sample Oriented EVOware Limited Edition SP1 See Tecan documentation on page 149 for a list of Tecan manuals detailing required parts and configuration The 100 mL troughs were purchased from Tecan Durham NC and the 1 5 mL tubes were purchased from Ambion Austin TX Prior to starting the validation the Freedom EVO 150 Liquid Handling workstation was installed and calibrated by Tecan The recommended maintenance schedule see the HID EVOlution qPCR STR Setup System Application Manual was followed e Quantification The Tecan Freedom EVO 150 Liquid Handling Workstation was used to set up the real time PCR reaction plates according to the protocol outlined in the Quantifiler Kits User Guide For PCR amplification up to three 7500 instruments were used all running the SDS Software v 1 2 3 A 77500 Results file was generated after the analysis using the 7500 SDS Real Time Software export tool then imported into the HID EVOlution M Software for use during STR PCR amplification setup Amplification The Tecan Freedom EVO 150 Liquid Handling Workstation was used to set up the STR PCR reaction plates according to the kit specific protocols described in the individual AmpF STR PCR Amplification kit user guides Positive amplification controls were manually diluted prior to amplification setup and the aliquot placed in the appropriate well of the reagent block After importing the 7500 R
57. If it is the last run of the day dispose of the TE buffer trough IMPORTANT Do not reuse the reagents in the troughs See Waste disposal on page 144 Follow the instructions in the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 1 After Run on page 87 HID EVOlution qPCR STR Setup System Getting Started Guide 35 Chapter 4 Run Automated qPCR Setup About the qPCR reaction plate layout About the qPCR reaction plate layout 36 Regardless of the extracted DNA sample setup the qPCR reaction plate is always set up in the same way the sample from the first position in the extracted DNA sample plate or first extracted DNA sample tube is always placed in well A3 of the qPCR reaction plate The DNA standard dilution series reactions are placed in columns 1 and 2 See Figure 3 In combined qPCR reaction plates the first Quantifiler Human sample reaction is placed in well A3 and the first Quantifiler Y Human Male sample reaction is placed in well A9 The DNA standard dilution series reactions are placed in columns 1 2 7 and 8 See Figure 4 on page 37 At the end of a qPCR reaction setup run the HID EVOlution gPCR STR Setup System generates a report that lists the position of each DNA sample in the extracted DNA sample plate or tubes and in the qPCR reaction plate 1 2 3 4 5 6 7 8 9 10 11 12 Figure 3 Plate layout for
58. Kit plus 2 or 3 controls per run See Before each run Set up extracted DNA samples on page 13 c Transfer the STR PCR reaction plate to the 9600 9700 or Veriti thermal cycler and perform PCR amplification HID EVOlution qPCR STR Setup System Getting Started Guide 91 Appendix C HID EVOlution gPCR STR Setup System Detailed Workflow Description 4 Perform capillary electrophoresis genetic analysis to separate and detect amplified STR products Prepare the CE plate Import the CE Setup file to the Data Collection Software v3 0 Run CE ona 3130 3130x Genetic Analyzer Note Any AB HID validated CE platform can be used but the electronic CE Setup file was formatted and validated for Data Collection Software v3 0 for the 3130 3130x Genetic Analyzers 5 Perform data analysis to analyze the CE results with GeneMapper D X Software or GeneMapper JD Software v3 2 92 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix D Dilution Protocols This appendix covers DNA input amount must be normalized before STR PCR amplification 94 Terms used in the Freedom EVOware software 0 00 00 eee 94 Guidelines for successful normalization 0 0 eee eee eee eee 94 Limits to the HID EVOlution system sample normalization 95 About DNA normalization on the HID EVOlution system 96 How the HID EVOlution system determines when to dilute an
59. Olution gPCR STR Setup System Getting Started Guide Chapter 3 Prepare qPCR Reagents and Labware 3 Set up reagents Set up reagents About minimum The tables on page 26 through page 28 list the minimum required reagent volumes for required reagent each kit The volumes in the tables include volumes p The volume of reagent that will be added to each reaction in the qPCR reaction plate Excess volume required per sample and per run necessary to compensate for evaporation and pipetting losses during the run Excess volume dead volume required per tube 50 uL or trough 5 mL on the worktable necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips For the Quantifiler Primer Mix and PCR Reaction Mix the volume required per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For these reagents the instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL this is 190 uL plus a 50 uL dead volume When the reagent volume falls below 240 uL the instrument is likely to determine that the liquid level is too low for successful aspiration If the instrument determines that the liquid levelis too low the instrument then looks for a sufficient volume in the next available tube of the same reagent if available or pauses the run and dis
60. R Reagents and Labware E 5 Check the extracted DNA samples a Ensure that the extracted DNA is in either a 96 well plate or in 1 5 mL tubes before setting up the samples on the worktable for STR PCR reaction setup For instructions see Set up extracted DNA samples in a 96 well plate on page 14 or Set up extracted DNA samples in tubes on page 15 b Confirm that there is an adequate volume of each extracted DNA sample IMPORTANT While the maximum sample volume required for any kit is 20 uL it is recommended that you check the samples for sufficient volume prior to dilution A 50 uL total volume is ideal for reliable liquid detection but you can use lower volumes If there is not enough sample for the required volume the entire volume will be aspirated and diluted according to the dilution protocol regardless of the deficient volume This deficiency could result in low or no profiles in the downstream analysis 6 Ifthe extracted DNA is in a plate place the extracted DNA sample plate into the metal plate adapter on the worktable in grid 21 site position 1 with well A1 in the top left corner Reaction Reaction plate 2 3 4 5 6 7 8 9 1 1 t Ge pae well 1 Y notch A1 ETSI ON CD SE Ee eff e2 oof foo sea ovo SEEIEJEIEEIEICIEIETERCI ET es IMPORTANT To ensure that samples are transferred to the correct wells confirm that The extracted DNA plate is placed in the metal plate adapt
61. R STR Setup file containing sample names and information was created then imported to the HID EVOlution qPCR STR Setup System software for use in qPCR reaction setup The 7500 Setup file generated by the HID EVOlution software output in txt format was used as the 7500 Setup file input for the 7500 instrument to perform qPCR The SDS software performed data analysis and produced a 7500 Results file output in csv format The qPCR STR Setup file containing sample names and information that was created in step 1 was imported to the HID EVOlution qPCR STR Setup System software for use in STR PCR amplification reaction setup The 7500 Results file output was used as the 7500 Results file input to dilute and normalize the samples on the HID EVOlution system All samples with concentrations lt 0 1 ng uL 0 025 ng uL were identified by the HID EVOlution system to receive 10 uL of DNA extract The HID EVOlution system generated a CE Setup file output in txt format and a STR Samples report The samples were then prepared for AmpF STR Identifiler kit amplification on the 9700 thermal cycler The CE Setup file output generated by the HID EVOlution software step 5 was imported to create a plate record into the 3130 3130x Genetic Analyzer Data Collection software for capillary electrophoresis fragment analysis Figure 39 on page 127 shows the steps used during HID EVOlution system check and
62. Setup file generated by the HID EVOlution qPCR STR Setup System for the CE instrument 1 After STR PCR reaction setup use Microsoft Excel to open the CE Setup file generated by the HID EVOlution qPCR STR Setup System Note The CE Setup file name and location is C HIDEVOlution_qPCRSTRfiles AB3130Input STRplate_ lt rundate gt _ lt runtime gt txt or C HIDEVOlution_qPCRSTRfiles AB3 130Input lt barcode gt _ lt rundate gt _ lt runtime gt txt 2 In the CE Setup file note the exact name including capitalization of the instrument protocol and the results group B C D E FP G H J K E M N Container IDescriptior ContainerT AppType Owner Operator 2 AS045GIE 20090123 96 Well Regular No log in No log in 3 AppServer Applnstance 4 GeneMapr GeneMapper Generic Instance 5 Well Sample Nz Comment Priority Sample Ty Snp Set Analysis lv Panel User Defin Size Stanc User Defin User Defih Results Group 1 Instrument Protocol 1 6 A1 50ng1 50ng1 100 Sample None HID_Advar MiniFiler 50ng1 HIDEvolution HIDEvolution 7 B1 TE1 TE1 100 Sample None HID_Advar MiniFiler TE1 HIDEvolution HIDEvolution 8 C1 50ng2 50ng2 100 Sample None HID_Advar MiniFiler 50ng2 AUD i HID i L9 D1 TE2 TE2 100 Sample None HID_Advar MiniF iler TE2 HIDEvolution HIDEvolution 10 E1 50ng3 5O0ng3 100 Sample None HID_Advar MiniFiler 5 ng3 HIDEvolution HIDEvolutio
63. Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 Running a Script for details Barcodes on tubes The sample name barcode and sample position for each tube are automatically updated in the software when the barcodes are scanned After the tube barcodes are scanned you have the option to manually edit the sample information See the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 Running a Script for details Import the qgPCR STR Sample file output from HID EVOlution Extraction System If you used the HID EVOlution Extraction System to perform DNA extraction use the Extraction System output qPCR STR Sample file HID_ lt rundate gt lt runtime gt csv from that run Sample ID and sample position in the sample file must agree with the samples on the worktable HID EVOlution gPCR STR Setup System Getting Started Guide 17 2 Chapter 2 Pre Run Procedures Before each run Optional Set up sample information Create a qPCR STR Sample file 18 T Create a gPCR STR Sample file from a template You can create a q PCR STR Sample file before running the script and then import the file into the HID EVOlution system Two sample input file templates are provided on the CD with the HID EVOlution software one for DNA samples in a 96 well plate and one for DNA samples in 1 5 mL tubes See Create a qPCR STR Sample file on page 18 and
64. ace and Appendix G Safety on page 141 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical WARNING Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices fe WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury fs WARNING Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Applied Biosystems instruments The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are av
65. action mix 13 Empty 5 mL VWR tube for the master mix 14 Quantifiler Human DNA Standard tube t The required number of tubes with the Quantifiler Y Human Male Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 uL plus a 50 pL dead volume If the volume of reagent in the first tube position 9 falls below 240 pL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid HID EVOlution qPCR STR Setup System Getting Started Guide 79 08 epino peuejs Buje uiejsAs dnjes H LS HOdb UONNIOAT AIH Quantifiler Human and Y Human Male Kits with pre prepared standards Quantifiler Human kit reagents Quantifiler Y Human Male kit reagents Figure 12 Reagent block configuration for Quantifiler Human and Y Human Male kits with pre prepared DNA standards Legend 1 8 1 5 mL tubes of pre prepared DNA standards arrange concentrations as shown in the corresponding orange tables 9 11 Up to three 1 5 mL tube s of Quantifiler Primer Mix Place the first tube in position 9 If you use more than one tube continue to position 10 then position 11 12 Q
66. age was 1144 RFU 474 Genotype concordance was 100 All samples with input amounts of 1 0 ng of DNA DNA starting concentrations of 0 1 ng uL resulted in a peak height ratio of 70 The variance in peak height averages was lt 16 between source vessels In conclusion sample dilution and normalization was successful regardless of the source vessel and starting DNA concentration for DNA concentrations 0 1 ng uL The extracted DNA samples with a starting concentration of 0 025 ng uL were not diluted 10 uL of DNA was directly transferred to the PCR reaction plate Samples with approximately 0 25 ng of DNA added to each PCR reaction displayed expected peak height averages that were smaller than the other samples due to the reduced mass of DNA The average peak height for reactions prepared from 0 025 ng uL DNA samples was 332 69 for samples in plates and 391 86 for samples in tubes The combined peak HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Precision and reproducibility studies STR PCR amplification reaction setup scripts height average for 0 25 ng input DNA samples in plates and tubes was 362 27 which is 29 of the average peak height for reactions with approximately 1 00 ng of input DNA DNA starting concentrations of 0 1 ng uL This result is consistent with the reduced amount of input DNA In conclusion the direct transfer of extracted DNA samples dur
67. ailable at www fbi gov hq lab fsc backissu july2000 codispre htm 134 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix F Automation Guidelines This appendix contains general guidelines to assist you with automating qPCR reaction setup DNA normalization and STR PCR amplification reaction setup on other liquid handling platforms E qPCR reaction setup automation guidelines sels 135 m STR PCR reaction setup automation guidelines lllillseuseu 137 qPCR reaction setup automation guidelines Before the automated process Prepare a DNA standard dilution series optional Prepare the reactions The following are general guidelines for automating the preparation of DNA standards and PCR reaction Master Mix and the aliquoting and mixing the Reaction Mix standards and samples for the Quantifiler reaction setup assays Prepare reagents and standards as described in the Quantifiler and AmpF STR kits 1 Ifusing plates be sure to arrange samples in a way that is compatible with the system 2 If preparing standards prepare T 4E buffer 10mM Tris HCl pH 8 0 0 1 mM Na EDTA 20 pg mL glycogen optional Prepare a DNA standard dilution series according to recommendations in the Quantifiler Kit Users Manual Calculate the volume of each component needed to prepare the reactions based on the following volumes e Quantifiler Human Primer Mix or Y Human Male Primer Mix
68. ailable to you free 24 hours a day For instructions on obtaining MSDSs see Obtaining MSDSs on page 143 IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer About This Guide How to obtain support How to obtain support For HID support e e In North America send an email to HIDTechSupport appliedbiosystems com or call 888 821 4443 option 1 Outside North America contact your local support office For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 1 The HID EVOlution gPCR STR Setup System Chapter 1 This chapter covers The HID EVOlution m HID EVOlution qPCR STR Setup System workflow 00 2 LI mR B Supported system configuration 20 00 c cece eee 4 B Supported kits ise ees Ses ee ele BEE AE A
69. ails Before starting the run if then run It is the first run of the day DailyStartUp qPCRSTR It is not the first run of the day Flush qPCRSTR When you run DailyStartUp qPCRSTR or Flush qPCRSTR one or more times until Fush RS Rh you sec e There are no visible air bubbles Air bubbles in the lines and and or e Flow from the DiTi cones is constant e Intermittent flow from a DiTi cone There are one or more DiTis on the liquid Drop DiTis qPCRSTR handling arm LiHa You refilled DiTis Set 200tip Position qPCRSTR and Set 50tip Position qPCRSTR IMPORTANT For proper liquid handling run the Flush qPCRSTR script before every run one or more times until there are no visible air bubbles Note Maintenance scripts are not included with Freedom EVOware v1 4 12 HID EVOlution qPCR STR Setup System Getting Started Guide Before each run Set up extracted DNA samples Chapter 2 Pre Run Procedures Before each run Set up extracted DNA samples Table 4 Maximum number of samples in a qPCR reaction setup run You can set up extracted DNA samples in either A 96 well plate Follow the instructions in Set up extracted DNA samples in a 96 well plate on page 14 1 5 mL tubes Follow the instructions in Set up extracted DNA samples in tubes on page 15 Note You cannot use both a plate and tubes for extracted DNA samples in the same run Tables 4 and 5 describe the maxim
70. al AmpF STR MiniFiler 4374618 Includes information about normalization and PCR Amplification Kit amplification specific to your kit User s Manual AmpF STR Profiler 4303501 Includes information about normalization and Plus PCR Amplification amplification specific to your kit Kit User s Manual AmpF STR SEfilerPlus 4385739 Includes information about normalization and PCR Amplification Kit amplification specific to your kit User s Manual AmpF STR SGM Plus 4309589 Includes information about normalization and PCR Amplification Kit amplification specific to your kit User s Manual AmpF STR Yfiler PCR 4358101 Includes information about normalization and Amplification Kit User s amplification specific to your kit Manual 3130x Getting Started 4352715 Includes information about using the 3130x Guide genetic analyzer GeneMapper ID v 3 1 4357520 Includes information specific to AmpF STR data User Guide analysis GeneMapper D Software 4352543 Includes detailed information on the features and Version 3 2 User Bulletin capabilities of GeneMapper D Software version 3 2 including support of the AmpF STR Yfiler PCR Amplification Kit 7300 7500 Real Time 4347828 Includes information on the 7500 System and PCR System Installation and Maintenance Guide SDS Software v1 2 3 HID EVOlution qPCR STR Setup System Getting Started Guide documentation Documentation Related documentation R
71. ar clothing and gloves when handling reagent and waste bottles A WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 143 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal HID EVOl
72. arch No right under any other patent claims such as apparatus or system claims for real time PCR is conveyed expressly by im plication or by estoppel Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS Trademarks of Life Technologies Corporation and its affiliated companies AB Design ABI PRISM AmpF STR COfiler FAM GeneAmp GeneMapper GeneScan Hi Di Identifiler LIZ MicroAmp MiniFiler PrepFiler Profiler Plus Quantifiler ROX SEfiler SEfiler Plus SGM Plus Veriti VIC and Yfiler AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc Freedom EVO and Freedom EVOware are registered trademarks and HID EVOlution is a trademark of Tecan Group Ltd All other trademarks are the sole property of their respective owners 2009 2010 Life Technologies Corporation All rights reserved Part Number 4426903 Rev B 09 2010 Chapter 1 Chapter 2 Chapter 3 Chapter 4 HID EVOlution gPCR STR Setup System Getting Started Guide Contents ADOUL Ihis Guide eurn uui uIOE RE PITE EMURS RS Paare iw esse ek vii Purpose rcx kms edhe eee kde RERAG eae oe be eet oh ae a 6E vii ASSUMPTIONS sx Lean ex UR ba ended beds dw a Seve eee dade Sele ees viii Safety information 000 ce eee ix How to obtain support
73. be 567 pL in one tube 10 12 uL reaction x 88 2 4 reactions 50 uL tube 1084 uL in one tube If you divide the minimum volume of PCR Master Mix between two tubes make sure that each tube contains a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For example place at least 620 uL in the first tube and 515 pL in the second tube seuinjoA jueDea euiuueje g eremqe pue sjueBeeu YOd HIS eedejg 9 Je1deuo LS t Includes excess volume to compensate for evaporation and pipetting losses during the run S A 50 uL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips If necessary combine reagents from different tubes from the same lot to meet the minimum volume requirements When ordering kits you can request multiple kits from the same lot tt 12 1 pL reaction x 88 2 0 4 reactions 50 uL tube 1143 84 uL in one tube S8 23 1 uL reaction x 88 2 0 4 reactions 50 uL tube 2138 24 uL in one tube If you divide the minimum volume of PCR Reaction Mix between two tubes make sure that each tube contains a multiple of 190 uL plus an additional 50 pL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For example place at least 11
74. be in position 9 If you use more than one tube continue to position 10 then position 11 12 Quantifiler PCR Reaction mix 13 Empty 5 mL VWR tube for the master mix t The required number of tubes with the Quantifiler Y Human Male Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 uL plus a 50 pL dead volume If the volume of reagent in the first tube position 9 falls below 240 pL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid 78 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix B Reagent Block Configurations Quantifiler DNA Quantification Kits Quantifiler Y Human Male Kit with system prepared standards Figure 11 Reagent block configuration for Quantifiler Y Human Male kit with DNA standards prepared by the HID EVOlution system Legend 1 8 1 5 mL empty tubes for DNA standards dilution series 9 11 Up to three 1 5 mL tube s of Quantifiler Y Human Male Primer Mix Place the first tube in position 9 If you use more than one tube continue to position 10 then position 11 12 Quantifiler PCR Re
75. bes make sure that each tube contains a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For example place at least 1000 uL in the first tube and 240 uL in the second tube You must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors seuinjoA jueDea euiuueje g eremqe pue sjueBeeu YOd HIS eedejg 9 Je1deuo Table 9 Yfiler kit reagent volumes 25 pL reaction volume for STR PCR amplification 6v t Includes excess volume to compensate for evaporation and pipetting losses during the run A 50 uL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips 5 5 uL reaction x 87 3 3 reactions 50 uL tube 561 5 uL in one tube tt 10 12 uL reaction x 87 3 3 reactions 50 uL tube 991 16 uL in one tube If you divide the minimum volume of PCR Reaction Mix between two tubes make sure that each tube contains a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For example place at least 810 uL in th
76. can use to import sample information during the qPCR and STR l PCR setup runs as described in Create a qPCR STR Sample file on page 18 O If you want the HID EVOlution qPCR STR Setup System to prepare DNA standards prepare the T E buffer 10 mM Tris HCI pH 8 0 and 0 1 mM Na EDTA according to the directions in the Quantifiler Kits User s Manual IMPORTANT Glycogen was not used during the Applied Biosystems validation study If you intend to use glycogen perform your own validation studies to evaluate the liquid handling performance Assemble the materials you will use See Required instruments software and materials on page 6 for a complete list of materials and sources e Extracted DNA samples as described in Set up extracted DNA samples in a 96 well plate on page 14 or Set up extracted DNA samples in tubes on page 15 e Reagents Quantifiler Human DNA Quantification Kit and or Quantifiler Y Human Male DNA Quantification Kit TgEo4 buffer 10 mM Tris HCI pH 8 0 and 0 1 mM Na EDTA Degassed deionized water system liquid 3000 mL per run e Plasticware AMicroAmp Optical 96 Well Reaction Plate used as the qPCR reaction plate f you want the HID EVOlution qPCR STR Setup System to prepare DNA standards eight labeled 1 5 mL tubes PN AM12400 or equivalent for DNA standards 16 tubes for combined setups One 5 mL VWR tube for the Quantifiler kit master mix 2 tubes for combined
77. cing the reagents in the reagent block E Quantifiler DNA Quantification Kits sss 76 Quantifiler Human Kit with pre prepared standards 76 Quantifiler Human Kit with system prepared standards T Quantifiler Y Human Male Kit with pre prepared standards 78 Quantifiler Y Human Male Kit with system prepared standards 79 Quantifiler Human and Y Human Male Kits with pre prepared standards 80 Quantifiler Human and Y Human Male Kits with system prepared standards 81 B AmpF STR PCR Amplification Kits losses 82 AmpF STR COfiler PCR Amplification Kit llis sees 82 AmpF STR Identifiler PCR Amplification Kit 08 83 AmpF STR MiniFiler PCR Amplification Kit 005 84 AmpF STR Profiler Plus PCR Amplification Kit 0 85 AmpF STR SEfiler Plus PCR Amplification Kit 00 86 AmpF STR SGM Plus PCR Amplification Kit 0 00005 87 AmpF STR Yfiler PCR Amplification Kit sceees ee 88 HID EVOlution qPCR STR Setup System Getting Started Guide 75 Appendix B Reagent Block Configurations Quantifiler DNA Quantification Kits Quantifiler DNA Quantification Kits Quantifiler Human Kit with pre prepared standards Figure 8 Reagent block configuration for Quantifiler Human kit with pre prepared DNA standards Legend 1 8 1 5 mL tubes
78. dard tube t The required number of tubes with the Quantifiler Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 pL plus a 50 uL dead volume If the volume of reagent in the first tube position 9 falls below 240 uL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid suoneinByuoo xoojg juebeey g xipueddy Syy UOMBOYNUEND YNG s ejnueno Bi Appendix B Reagent Block Configurations AmpF4STR PCR Amplification Kits AmpF STR PCR Amplification Kits AmpF STR COfiler PCR Amplification Kit Figure 14 Reagent block configuration for COfiler kit Legend 1 Control DNA 9947A diluted as necessary See step 4 on page 52 2 1 5 mL tube containing T 5E buffer 3 Empty VWR tube for master mix preparation 4a 4b AmpF STR PCR Reaction Mix 5 COfiler Primer Mix 6 AmpliTaq Gold DNA polymerase t You must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTag Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection err
79. delines STR PCR reaction setup automation guidelines 140 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix G Safety This appendix covers NH Chemical safety sesto Gar oen eae eae eae aed 142 General chemical safety 0 nnana ccc cc eens 142 NISDSS 445 455 Re SCC S RODENT A ed Vaid udi 143 Chemical waste safety 0 eee teens 144 Biological hazard safety 0 0 cece cece eee eee 145 B Safety alerts duo pee ei ERU Rex SEDE RAE ee eee 146 HID EVOlution qPCR STR Setup System Getting Started Guide 141 e Appendix G Safety Chemical safety Chemical safety General chemical safety 142 Chemical hazard WARNING CHEMICAL HAZARD Before handling any chemicals refer to warning the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions d WARNING CHEMICAL HAZARD AII chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewe
80. e Positive Amplification Control 124 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Identifiler and SGM Plus STR PCR amplification reaction setup accuracy study Table 21 Summary of peak height averages for SGM Plus from samples n 11 with different starting DNA concentrations before normalization diluted and prepared either manually or with the HID EVOlution system automated Peak Height Averages RFU Concentration Mana RFU Manua Ae Ate yamaha Petite 0 025 234 13 278 13 8496 0 1 1000 70 983 80 1093 50 1066 174 92 0 5 1139 10 1412 90 81 1 992 80 95230 10496 2 956 40 963 10 9996 5 947 00 989 20 9696 10 977 40 1146 80 8596 25 874 30 90440 9796 50 234 13 278 13 8496 HID EVOlution qPCR STR Setup System Getting Started Guide 125 E Appendix E Validation Experiments and Results Complete system check and precision study Complete system check and precision study Experiment To evaluate the precision of the entire HID EVOlution system workflow genomic DNA was diluted with T E buffer to obtain concentrations of 50 0 10 0 5 0 2 0 1 0 0 5 and 0 025 ng uL Ten replicates of each concentration were quantified 80 samples in total using the Quantifiler Human kit The quantification standards were prepared by the HID EVOlution system 1s A qPC
81. e including running the appropriate maintenance scripts according to the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 4 3 2 Prepare the Instrument section 5 1 Starting the System and Before each run Run maintenance scripts on page 12 in this guide Determine the required volume of reagents for the run as described in Determine reagent volumes on page 47 Place the reagents in the reagent block as described in Set up reagents on page 52 OOO Place the reagents labware and samples on the worktable as described in Set up the labware on the worktable on page 54 46 HID EVOlution gPCR STR Setup System Getting Started Guide Determine reagent volumes Chapter 6 Prepare STR PCR Reagents and Labware E Determine reagent volumes About minimum Tables 8 through 11 on pages 48 through 51 list the minimum required reagent required reagen volumes for each kit The volumes in the tables include volumes The volume of reagent that will be added to each reaction in the STR PCR reaction plate Excess volume required per sample and per run necessary to compensate for evaporation and pipetting losses during the run Excess volume dead volume required per tube 50 uL or trough 5 mL on the worktable necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips For the AmpF STR Prime
82. e first tube and 240 uL in the second tube You must combine the volumes in two full 50 pL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors I cO m 5 Required Extracted Control Required Required E Available Volume DNA Samples Excess Dead o Reagent Volume in per Samples er ES Reactions Volume m E 9 Full Tube Reaction per Run p per Run per Tube Minimum Required Volume for E of Reagent 88 Samples and 2 Controls d A B C D E S AmpF STR Yfiler Primer Set 0 55 mL 5 5 uL up to 87 3 3 50 uL A x B C D E X 562 pL Co 2 AmpF STR Yfiler PCR Reaction 1 1 mL 10 12 uL up to 87 3 3 50 uL A x B C D E E ME 992 uL in one tube E AmpliTag Gold DNA Polymerase 50 uL 100 uL combined in one tube for 1 to 87 samples plus 3 controls 100 uL combined in one tube 3 D AmpF STR Control DNA 9947A 25 uL 60 uL for 1 to 87 samples plus 3 controls 60 uL F AmpF STR Control DNA 007 300 uL 60 uL for 1 to 87 samples plus 3 controls 60 uL Q Q T40Eo 1 buffer in reagent block NA 60 uL for 1 to 87 samples plus 3 controls 60 uL fad _ g T4o9Eo 4 buffer in trough NA 25 mL in trough for 1 to 87 samples plus 3 controls 25 mL in trough Q c Qo oO eremqe pue sjueBeeu Hod HIS aedaly 9 Je1deuo seuinjoA u be euiuue
83. e results group in the Data Collection software Results Group Editor a Select the General tab then enter the results group name exactly as it appears in the CE Setup file usually HIDEvolution b Select the Analysis tab then select GeneMapper Generic in the Analysis Type field c Configure the Destination and Naming tabs according to your laboratory needs 11 2 Chapter 2 Pre Run Procedures Before each run Run maintenance scripts 5 If necessary set up the instrument protocol in the Data Collection software a Select Protocol Manager from the left navigation pane b Click New at the center left of the Instrument Protocol section then enter the following Name Enter the instrument protocol name exactly as it appears in the CE Setup file usually HIDEvolution Type Regular Run Module Select the run module according to the kit and your laboratory needs DyeSet Select the dye set according to the kit c Click OK Refer to the Applied Biosystems 3 30 3130xl Getting Started Guide for details on creating the instrument protocol and results group Before each run Run maintenance scripts Run the appropriate maintenance scripts after setting up carriers and racks on the worktable but before placing samples reagents or plasticware on the worktable See the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 7 5 Maintenance Scripts and Section 5 2 Running Maintenance for det
84. e v3 0 Create detectors If you have not already done so create detectors in the SDS Software v1 2 3 for forthe 7500 Real running the Quantifiler assays Refer to the Applied Biosystems Quantifiler Kits Time PCR System 56 Manual for instructions on creating the detectors IMPORTANT The detectors must have the exact names and capitalization shown below Note These detectors allow you to import the HID EVOlution system generated 7500 Setup file ReactionPlatel txt or lt barcode gt txt to the SDS Software See About the 7500 Setup file on page 40 for details 1 Create the following detectors for the Quantifiler Human kit Name Quantifiler Human Reporter Dye FAM Quencher Dye None make sure None is selected Name IPC IPC assay Reporter Dye VIC Quencher Dye None make sure None is selected 2 Create the following detectors for the Quantifiler Y Human Male kit Name Quantifiler Y Reporter Dye FAM Quencher Dye None make sure None is selected Name IPC IPC assay Reporter Dye VIC Quencher Dye None make sure None is selected 10 HID EVOlution qPCR STR Setup System Getting Started Guide Create an instrument protocol and results group Chapter 2 Pre Run Procedures P One time tasks Ifyou have not already done so create an instrument protocol and a results group in the Data Collection Software v3 0 to match the names used in CE
85. eave empty positions between sample tubes IMPORTANT DNA sample tubes must be contiguously loaded Do not leave empty tube positions between sample tubes Examples of correct qPCR setup e 6 e e e C O C e e C e D O e 606066600009 0500 Front of instrument 15 2 Chapter 2 Pre Run Procedures Before each run Set up extracted DNA samples Examples of correct STR PCR setup e OO C2 eeooo oo eooo ClO O ecOoOoO OO O eooo ClO T eooo ClO O OIOIOO Cle OIOOLO OO e DOCG OO O ACCC Clo O OOOO Clo O OOOO Cle O OOOO Cle O OOOO Cle O OCOOO Cle z OO E ECIAM Front of instrument 4 Check that the barcodes are in a readable position 5 Open each tube securing the tube caps in a fixed upright position as shown below IMPORTANT Open tube caps carefully to prevent contamination and splatter HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 2 Pre Run Procedures 2 Before each run Optional Set up sample information Before each run Optional Set up sample information You have several options for entering sample information to the HID EVOlution qPCR STR Setup System software If you chose the sample file option set up the sample file before the run according to the instructions in Create a qPCR STR Sample file on page 18 About sample During a qPCR or STR PCR reaction setup run the software prompts you to
86. efer to the Tecan documents in the following table for details relating to the Tecan Freedom EVO instrument and software and the HID EVOlution system References to the appropriate Tecan documentation are also provided throughout this guide For information on Refer to The HID EVOlution system application Tecan HID EVOlution qPCR STR Setup System Application Manual PN 394918 Installing and setting up the Freedom EVO instrument Tecan HID EVOlution Installation Manual Tecan Software Manual Instrument Software V6 1 Part 1 Freedom EVOware Getting Started Guide Comprehensive safety information operating maintenance and troubleshooting procedures for the Freedom EVO instrument Tecan Freedom EVO Operating Manual PN 392886 Tecan Freedom EVO Maintenance and Service Logbook PN 392185 Tecan Freedom EVO Daily Weekly Maintenance Checklist PN 392818 I Installing setting up running and programming the Freedom EVOware software Tecan Freedom EVOware Standard 2 1 Plus 2 1 Software Manual Tecan Freedom EVOware Standard 2 1 Plus 2 1 Software Getting Started Guide Freedom EVOware Software Manual Extended Device Support Research Use Only PN 393172 Freedom EVOware Software Manual Limited Dev Support General Purpose PN 393804 j Running scripts and troubleshooting script related error messages Tecan Freedom EVOware Standard 2 1 Plus 2 1
87. ells for amplification positive control 1 well for amplification negative control 6 empty wellst t Because the STR PCR reaction plate layout is designed for easy transfer of amplified PCR product to a CE plate six of the wells in the STR PCR reaction plate remain empty as a placeholder for the allelic ladder sample replicates in the CE plate See About the STR PCR plate layout on page 62 HID EVOlution qPCR STR Setup System Getting Started Guide 13 2 Chapter 2 Pre Run Procedures Before each run Set up extracted DNA samples Set up extracted 1 DNA samples in a 96 well plate 14 Confirm that a MicroAmp Optical 96 Well Reaction Plate the extracted DNA sample plate is labeled for identification If you use barcodes to track samples move the barcode provided with the plate into the correct position as shown in the Tecan HID EVOlution qPCR STR Setup System Application Manual Place the first extracted DNA sample in any well position on the plate for example you can begin with well number 14 After the first extracted DNA sample continue placing samples next to one another in vertical columns as shown in the Correct examples below Do not leave empty wells between samples If there is a failed extracted sample in the plate use blank reagents water or TE buffer in that well See examples of correct extracted DNA sample plate setup below IMPORTANT Make sure to assign a sample ID to all samples inc
88. eptable for proper liquid handling and no control will be transferred to the STR PCR reaction plate 5 Select the appropriate reagent block setup from the following table then use the appropriate figure to place the reagents and empty tubes in the correct positions in the chilled reagent block Note There is a 1 5 mL tube of T E buffer placed in the Reagent block that is separate from the TE buffer placed in a trough See Set up the labware on the worktable on page 54 for instructions on placing the T 9E buffer trough on the worktable If you are using the following Set up the reagent block AmpF STR PCR Amplification kit according to COfiler kit Figure 14 on page 82 Identifiler kit Figure 15 on page 83 MiniFiler kit Figure 16 on page 84 Profiler Plus kit Figure 17 on page 85 SEfiler Plus kit Figure 18 on page 86 SGM Plus kit Figure 19 on page 87 Yfiler kit Figure 20 on page 88 HID EVOlution gPCR STR Setup System Getting Started Guide 53 Set up the labware on the worktable E Chapter 6 Prepare STR PCR Reagents and Labware Set up the labware on the worktable After loading the reagent block use the following procedure to place the reagent block STR PCR reaction plate trough disposable pipette tips DiTis and samples on the worktable Use Figure 5 on page 56 as a guide CAUTION For important safety information related to the use of the Tecan Freedo
89. equired N Available chine l DNA Standard Excess Dead Minimum Required Volume for Redden Volume in a fa Samples Samples Reactions Volume 32 extracted DNA samples 9 Full Tube of per Run per Run per Run per Tube and 32 diluted standards Reagent A x B C D E A B Cc D E Quantifiler Human Primer 1 4 mL 11 55 uL up to 32 16 3 50 uL 639 uL in one tube Mix Quantifiler Y Human Male 1 4 mL 11 55 uL up to 32 16 3 50 uL 639 uL in one tube Primer Mix Quantifiler PCR Reaction 5mL 13 75 uL up to 32 16 3 50 uL 752 uL in one tube Mix for preparing Quantifiler Human reactions Quantifiler PCR Reaction 5mL 13 75 uL up to 32 16 3 50 uL 752 uL in one tube Mix for preparing Quantifiler Y Human Male reactions A 50 uL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips If you divide the volume of Primer Mix across multiple tubes each tube must contain a multiple of 190 uL plus an additional 50 L per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions E sjueDeai dn jeg ejewqe pue sjusbeey uodgdb eredejd e Jeydeuo Chapter 3 Prepare qPCR Reagents and Labware 3 Set up reagents Place reagents in Use the following procedure to place the Quantifiler kit reagents into the correct the qPCR reagent positions in the chilled qPCR reagent block block 1 Thaw reagents mix and ce
90. er The extracted DNA plate wells are aligned with the holes in the metal plate adapter e Well AI is positioned in the upper left corner HID EVOlution qPCR STR Setup System Getting Started Guide 55 Chapter 6 Prepare STR PCR Reagents and Labware Set up the labware on the worktable 000000000000 000000000000 Site number 1 O0000000 Oo0000000 OOo oo oo Site number 2 O0000000 OO0000000 OO0000000 OO0000000 o o o o o o o o O0000000 O0000000 O0000000 O O O oou ood oOo mm 00 Oo0000000 O0000000 O0000000 O0000000 Poo OO Site number 3 O0000000 O0000000 O0000000 O0000000 oon O0000000 Oo0000000 O0000000 00000000 LO ooOoOmmo0o ood 000000000000 1 82 83 84 85 S6 Grid numbers 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 5 Tecan Freedom EVO workstation worktable layout for a STR PCR setup run Locations for DNA samples in a plate and tubes are shown Note Only one type of plasticware for extracted DNA can be placed on the workstation for a given run 1 2 200 pL disposable pipette tips DiTis 3 6 50 uL DiTis 7 Trough for T 9E 4 buffer 8 AB STR PCR Reagent Block 9 MicroAmp Optical 96 Well Reaction Plate if samples are in a plate with 96 well metal plate adapter 10 11 MicroAmp Optical 96 Well Reaction Plates for predilution of samples with 96 well metal plate adapter
91. esults file s into the HID EVOlution Software extracted DNA samples with concentrations lt 0 1 ng uL and negative controls were selected for direct transfer of the maximum allowable volume 10 or 20 uL into the STR PCR reaction plate Extracted DNA samples with concentrations between 0 1 ng uL and 50 ng uL were diluted according to the software parameters See Appendix D Dilution Protocols on page 93 for additional details about dilution protocols PCR amplification was carried out in calibrated 9700 thermal cyclers HID EVOlution qPCR STR Setup System Getting Started Guide 105 106 Appendix E Validation Experiments and Results Materials and methods Data analysis Capillary electrophoresis Capillary electrophoresis plates were prepared manually Master mixes of Hi Di Formamide and GeneScan 500 LIZ Size Standard GeneScan 500 ROX Size Standard for 50 uL reactions PCR product and kit specific allelic ladder were prepared and added manually to the samples according to the procedure described in each kit specific user guide The CE Setup file generated by the HID EVOlution Software during the STR PCR setup run was imported to the Data Collection Software v3 0 on a 3130x Genetic Analyzer to create a plate record Capillary Electrophoresis was performed on an ABI PRISM 3130x Genetic Analyzer with Data Collection Software v3 0 using recommended protocols For analysis of the Quantifiler kit result
92. ety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory 144 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix G Safety e Chemical safety Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety General biohazard AN WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals
93. fety Chemical safety Chemical waste safety Chemical waste CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and hazards local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines yg Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example saf
94. ged according to the specific kit used and shown in this guide and in the HID EVOlution qPCR STR Setup System Application Manual Freedom EVOware v2 1 was validated on the HID EVOlution Combination System with additional verification studies performed on the HID EVOlution qPCR STR Setup System The Applied Biosystems kits that were validated include the Quantifiler Human DNA Quantification kit Quantifiler Y Human Male DNA Quantification kit AmpF STR Identifiler PCR Amplification kit AmpF STR MiniFiler PCR Amplification kit AmpF STR Yfiler PCR Amplification kit AmpF STR SGM Plus PCR Amplification kit AmpF STR Profiler Plus Amplification kit AmpF STR COfiler PCR Amplification kit and AmpF STR SEfiler PCR Amplification kit The SEfiler Plus kit was validated separately the results are not presented in this chapter but are in line with the other kit validation results presented here For kit part numbers see Supported kits on page 5 The experimental data that were generated demonstrated that the HID EVOlution system v1 0 was functional and consistent in its ability to Produce accurate and reproducible results when used with the Quantifiler Human and Quantifiler Y Human Male kits Import 7500 Results files generated with SDS software v1 2 3 Normalize samples based on imported real time PCR results Provide accurate and reliable STR results when used with all supported Applied
95. ghten the DiTi cones diluter valves and syringe fittings Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual Confirm that you have sufficient system liquid then flush the system and check for air bubbles and leaks Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual CE signal is too high Sample concentration is greater than the reported quantification result Requantify see IMPORTANT above Before the next run To ensure correct pipetting clean and finger tighten the DiTi cones diluter valves and syringe fittings Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual Confirm that you have sufficient system liquid then flush the system and check for air bubbles and leaks Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual 72 HID EVOlution qPCR STR Setup System Getting Started Guide Table 12 Troubleshooting qPCR STR PCR results continued Appendix A Troubleshooting Observed problem Possible reason Suggested solution CE signal is too low Potential presence of PCR inhibitors Incorrect target amount entered into the Sample Normalization Adjustment window Not enough DNA Presence of inhibitors Degraded formamide Dilution of DNA sample in H O or wrong buffer e g wrong EDTA concentration Confirm reagent and instrument performance Ma
96. have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov HID EVOlution qPCR STR Setup System Getting Started Guide 145 e Appendix G Safety Safety alerts Safety alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page ix 146 HID EVOlution qPCR STR Setup Sy
97. hibited a Cy value of 38 This sample was amplified using MiniFiler and standard HID analysis methods It showed no STR profile The absence of a profile confirmed that the C4 value of 38 was a result of stochastic variation in the qPCR reaction and not due to a cross contamination of the sample during automated setup A separate set of source plates with the same source DNA as above and the same layout patterns was used to test the STR PCR setup operations for cross contamination The 80 wells containing TE 40 from each pattern were evaluated for profiles after fragment analysis This set of TE studies also showed no STR profile HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Conclusion Conclusion Automated protocols were developed for and validated on the HID EVOlution System for the following Quantifiler Human Quantifiler Y Human Male Identifiler Yfiler MiniFiler SGM Plus Profiler Plus SEfiler and COfiler kits Validation studies demonstrated that the liquid handling protocols provided robust and reliable results using a range of DNA quantities In conclusion the following observations were made e Quantification scripts Quantification scripts produced standard curve results that were within acceptable limits as defined by PCR efficiency and extensive Applied Biosystems experience manually preparing Quantifiler kits Variat
98. in a 96 well plate Peak height averages were calculated and compared for samples prepared on the HID EVOlution system and samples prepared manually For Identifiler the peak height variation was comparable to data generated from manually prepared samples see Figure 37 and Table 20 on page 123 The peak heights observed for samples with less than 0 1 ng uL had lower peak heights as expected and the reduction correlated with the DNA input amount HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Identifiler and SGM Plus STR PCR amplification reaction setup accuracy study Correlation Between Manual and Automated Operations 3000 2500 4 2000 Average Peak Height I e e o L ck Ell 0 f i als a a T UL T al EU XO GN RAED RA EME Mes errs Es On ON S USUS D OS LIP SS ero Q o 9 amp DUET Source DNA ng uL Figure 37 Average peak heights at varying starting DNA concentrations Results from Identifiler PCR amplification reactions prepared by the HID EVOlution system are shown in yellow and those prepared manually are shown in green Table 20 Summary of Peak Height Averages for Identifiler from samples n 11 with different starting DNA concentrations before normalization diluted and prepared either manually or with the HID EVOlution system automated Peak Height Averages RFU
99. ing Started Guide Appendix D Dilution Protocols Guidelines for successful normalization Limits to the HID The HID EVOlution system software uses the target amount of DNA to select the EVOlution system required dilution protocol It is not possible for the HID EVOlution system to dilute Ed exceptionally high concentrations of DNA to the smallest target amount Given a two step dilution process and a maximum volume of 200 uL per dilution step the maximum achievable dilution for a 1 ng target is 1 4000 for a 25 uL total reaction volume and 1 8000 for a 50 uL total reaction volume In the Sample Normalization Adjustment window if you select a sample that requires more than a 1 4000 dilution or 1 8000 dilution for a 50 uL reaction volume the sample will only be diluted by 1 4000 or 1 8000 respectively After dilution HID EVOlution system will place the maximum transferable volume either 10 or 20 uL into the STR PCR reaction plate Consequently the HID EVOlution system may add too much DNA sample to the STR PCR plate This could result in an off scale DNA data profile Table 14 lists the maximum and minimum concentrations that can be diluted to the specified target amount for either a 10 uL or 20 uL transfer Use Table 14 to determine the maximum concentration that can be diluted for the target DNA input amount ng For example the HID EVOlution system cannot dilute a 1000 ng uL sample to achieve a 0 1 ng target input amount
100. ing automated Identifiler assay setup using a concentration less than the desired target input amount was successful regardless of whether the samples started in a plate or in a tube 4000 3000 Maia i 0 025 0 1 0 5 1 0 2 0 5 0 10 0 50 0 Source DNA ng uL Average Peak Height RFU 04 Figure 27 Identifiler kit precision and reproducibility studies depicting the average peak height distribution of each study Samples were present either in plates green or in tubes yellow Studies were carried out on separate days each represented by a separate box plot in the appropriate color Figure 28 on page 116 is data from the SGM Plus STR PCR setup The SGM Plus kit used a 50 uL PCR reaction volume with a 20 uL volume for normalized DNA diluted as necessary The overall peak height average for all experiments using the SGM Plus kit regardless of the source vessel was 938 RFU with a standard deviation of 354 not including the 0 025 ng samples For tubes the peak height average was 958 RFU 344 For plates the peak height average was 919 RFU 362 Genotype concordance was 100 and all samples with input amounts 0 1 ng uL resulted in a peak height ratio of 2 7096 HID EVOlution qPCR STR Setup System Getting Started Guide 115 116 Appendix E Validation Experiments and Results Precision and reproducibility studies STR PCR amplification reaction setup scripts
101. ing tubes of reagents 52 configured volume in Sample Normalization window 94 contamination guidelines for preventing 20 control DNA preparing diluted 52 cooling reagent block 24 46 create detectors for 7500 Real Time PCR System 10 instrument protocol 11 qPCR STR Sample file 18 results group 11 customer feedback on Applied Biosystems documents 150 D DANGER description ix diluting control DNA 52 dilution guidelines 94 original sample volume used in 98 protocol tables 99 protocols 93 Sample Normalization Adjustment window 59 dilution ratio determining 98 examples 99 DNA extraction procedures 2 DNA samples in a 96 well plate 14 intubes 15 DNA standard dilution series glycogen 24 volumes for pre prepared series 26 volumes for system prepared series 27 DNA yield troubleshooting absence of DNA 71 troubleshooting low volume 71 151 Index documentation Applied Biosystems 148 Tecan 149 E enter sample information in EVOware software 17 export 7500 Results file 42 extracted DNA in a 96 well plate 14 intubes 15 extraction procedures for 2 F file name and location 7500 Setup file 40 CE Setup file 67 forensic workflow overview vii 2 G glycogen in DNA standard dilution series preparation 24 guidelines chemical safety 142 chemical waste disposal 144 chemical waste safety 144 dilution 94 normalization 94 preventing contamination 20 H hazards See safety Help system accessing 150 HID EVOlut
102. ion Dilution Dilution Sample plate 1 2 DNA If sample concentration D1 If dilution ratio gt 1 20 D2 is greater than target 10 uL STR PCR plate 4 10 HL If sample concentration is lower than target 10 uL PCR Workflow for normalization using a 20 pL addition Dilution Dilution Sample plate 1 2 DNA If sample concentration D1 If dilution ratio gt 1 40 D2 is greater than target 20 uL STR PCR plate 4 20 HL If sample concentration is lower than target 20 uL PCR Figure 21 Normalization workflows 10 uL and 20 uL Samples with DNA concentrations lower than the target will not be processed unless you manually select these samples for processing in the Sample Normalization Adjustment window HID EVOlution qPCR STR Setup System Getting Started Guide 97 Order of processing and placement of D1 and D2 in the pre dilution plates Appendix D Dilution Protocols Dilution protocols with examples The HID EVOlution gPCR STR Setup System processes all of the samples that require a one step dilution first then processes all of the samples that require a two step dilution For two step dilutions the first D1 and second D2 dilutions occur in the same predilution plate in adjacent wells for example wells 41 and 49 or wells A6 and AT The two predilution plates loaded onto the worktable do not correspond to the number of dilutions a
103. ion Extraction System in HID workflow 3 using qPCR STR Sample file from 17 HID EVOlution qPCR STR Setup System configuration 4 detailed workflow 89 in HID workflow vii 2 instruments used with 6 materials required for use with 6 prerequisites viii sample information files 3 software used with 6 supported kits 5 Identifiler kit reagent block configuration 83 required reagent volumes 48 152 import 7500 Setup file to SDS software 41 CE Setup file to Data Collection software 68 sample information into EVOware software 17 59 IMPORTANT description ix Information Development department contacting 150 instrument protocol create 11 instrumentation for use with AmpF STR kits 6 for use with Quantifiler kits 6 IPC Cy troubleshooting 72 L labware for qPCR reaction setup 30 for STR PCR reaction setup 54 M maintenance scripts run 12 manually enter sample information 17 59 maximum samples per run qPCR reaction setup 13 STR PCR reaction setup 13 MiniFiler kit reagent block configuration 84 required reagent volumes 50 MSDSs about ix description 143 obtaining x 143 N normalization adequate sample volume for 94 examples 99 guidelines 94 limits 95 original sample volume used in 98 Sample Normalization Adjustment window 59 O online Help See Help system output file from qPCR reaction setup 40 from qPCR run 42 from STR PCR reaction setup 67 HID EVOlution gPCR STR Setup System
104. ion Import Files Export Files Extraction Sample File qPCR STR Sample File 7500 Setup File qPCR STR Sample File 7500 Setup File 7500 Results File s qPCR STR Sample File CE Setup File 7500 Results File s MEE CE Setup File CE Results Files Sample Profiles STR Results CE Results Files Figure 1 Files used to import sample information to the next HID workflow step The qPCR STR Sample file is automatically generated by the HID EVOlution Extraction System or can be manually created from a template Use the qPCR STR Sample file to import the required sample name and information for both qPCR and STR PCR reaction setup see page 17 for more options Import the automatically generated 7500 Setup file as a SDS plate record Import the automatically generated CE Setup file as a CE plate record HID EVOlution qPCR STR Setup System Getting Started Guide 3 1 Chapter 1 The HID EVOlution qPCR STR Setup System Supported system configuration Supported system configuration The HID EVOlution gPCR STR Setup System consists of A dedicated TECAN Freedom EVO 150 or 200 robotic workstation The necessary hardware including a 4 channel liquid handling accessory LiHa Application software Freedom EVO ware version 2 1 SP1 SOE version 1 2 SP1 and HID EVOlution qPCR STR driver 1 1 0 0 25 SP4 or Freedom EVOware version v1 4 SP1 special SOE for HID and HID EVOlut
105. ion 6 Results 37 4 Chapter 4 Run Automated qPCR Setup For more information 38 HID EVOlution qPCR STR Setup System Getting Started Guide The HID EVOlution qPCR STR Setup System Pre Run Procedures Prepare qPCR Reagents and Labware Run Automated qPCR Setup Chapter 5 Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis Chapter 5 Perform qPCR and Review Results This chapter explains where to get relevant information to perform qPCR and review the results B About the 7500 Setup file 0 cee ees 40 E Transfer and import the 7500 Setup file 2 2 0 eee 41 B Perform gPCR noneesdebr Set aud dd ave da Shanes deh DEINER and 42 E Analyze and export the qPCR results 0 20 0 ees 42 E For more information 0 000 cece e 43 HID EVOlution gPCR STR Setup System Getting Started Guide 39 H Chapter 5 Perform qPCR and Review Results About the 7500 Setup file About the 7500 Setup file The HID EVOlution qPCR STR Setup System software generates a 7500 Setup file for each run The file contains sample information that you can import to an SDS software plate document for the qPCR run on the 7500 Real Time PCR instrument By default the HID EVOlution qPCR STR Setup System names the 7500 Setup fi
106. ion between scripts employing either tubes or plates was minimal as was the variation between automation and manually prepared samples STR PCR amplification scripts STR PCR amplification scripts produced average peak height results that were within acceptable limits as defined by instrument specific parameters with results within the desired peak height range of 1000 3000 RFU Variation between scripts employing either tubes or plates was minimal as was the variation between automation and manually prepared samples Entire workflow Sample names and information were accurately tracked and reported throughout the workflow Quantification data was properly integrated and sample normalization was accurately defined STR electropherograms exhibited desired peak height averages intralocus balance and intracolor balance resulting in acceptable STR profiles HID EVOlution qPCR STR Setup System Getting Started Guide 133 E Appendix E Validation Experiments and Results References References Scientific Working Group on DNA Analysis Methods 2004 Revised Validation Guidelines approved July 2003 Forensic Science Communications Volume 6 Number 3 Available at www fbi gov hq lab fsc backissu july2004 standards 2004 03 standards02 htm DNA Advisory Board 2000 Quality Assurance Standards for Forensic DNA Testing Laboratories approved October 1998 Forensic Science Communications Volume 2 Number 3 Av
107. ion qPCR STR driver 1 1 0 0 25 SP4 Note If you are not using the most current driver 1 1 0 0 25 SP4 contact your local Tecan service organization about upgrading See the Tecan HID EVOlution gPCR STR Setup System Application Manual Section 10 Customer Support for contact information Software scripts developed and validated to automate the set up of reaction plates for quantitative real time PCR and STR PCR amplification for use in HID applications Note The Freedom EVO 150 and 200 instruments can be configured identically and both instruments are supported for use with the HID EVOlution qPCR STR Setup System Validation studies were performed on the Freedom EVO 150 and Freedom EVOware v1 4 Freedom EVOware v2 1 was validated on the HID EVOlution Combination System with additional verification studies performed on the HID EVOlution qPCR STR Setup System HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 1 The HID EVOlution gPCR STR Setup System 1 Supported kits Supported kits The following Applied Biosystems DNA quantitation and amplification kits have been validated and are supported for use with the HID EVOlution qPCR STR Setup System Supported Kit ABRET rine ll Quantifiler Human DNA Quantification Kit 4343895 Quantifiler Y Human Male DNA Quantification Kit 4343906 AmpF STR COfiler PCR Amplification Kit 4304256 AmpF STR Ide
108. is Import a CE plate record Chapter 8 Set up CE reactions Run CE Y Perform Data Analysis Figure 1 Overview of the automated HID workflow using the HID EVOlution qPCR STR Setup System The steps covered in this Getting Started Guide are shown in blue The steps performed on the HID EVOlution system are highlighted in orange vii About This Guide Assumptions Assumptions viii This guide assumes that You know how to handle forensic samples and prepare them for quantitation and STR analysis The HID EVOlution gPCR STR Setup System a Tecan Freedom EVO 150 or 200 with the appropriate hardware software and scripts has been installed configured tested and calibrated by Tecan personnel You are trained on the proper operation maintenance and troubleshooting of the HID EVOlution qPCR STR Setup System You have access to the Tecan HID EVOlution qPCR STR Setup System Application Manual the Tecan Freedom EVO Operating Manual and other applicable Tecan documentation You have referred to the manufacturer s instrument documentation for important safety information related to the use of the Tecan Freedom EVO instrument HID EVOlution qPCR STR Setup System Getting Started Guide About This Guide Safety information Safety information Safety alert words MSDSs HID EVOlution gPCR STR Setup System Getting Started Guide Note For general safety information see this Pref
109. is first dilution is only a 1 20 dilution To further dilute the sample the HID EVOlution system aspirates and mixes 22 uL of the D1 dilution with 18 uL of TE in the second dilution D2 The two dilution steps take place in adjacent wells of the same predilution plate The second predilution plate is required when a large number of samples are processed and or a large number of dilutions are required Finally the HID EVOlution system will aspirate and add 10 uL from D2 directly to the STR PCR Plate D2 to PCR Dilution protocol The maximum total dilution is 1 4000 for a 25 uL total reaction volume or 1 8000 for tables a 50 uL total reaction volume Below are two example dilution protocol tables that target 1 ng of DNA Table 15 is for the 5 dye kits that require 10 uL of diluted DNA 25 uL total reaction volume and Table 16 on page 101 is for the 4 dye kits that require 20 uL of diluted DNA 50 uL total reaction volume in the STR PCR plate Note The 10 and 20 uL dilution tables are example dilution protocols for a 1 ng target DNA input amount The dilution protocols used by the HID EVOlution system software will change based on the modifiable parameters such as the target DNA input amount see step 5 on page 59 See either the Sample Normalization Adjustment window or the STR Samples report for the specific dilution used for an individual sample HID EVOlution qPCR STR Setup System Getting Started Guide 99 Appendix D Dil
110. ity study on page 108 was repeated with male genomic DNA using the Quantifiler Y Human Male Kit Automated setup of The precision of the automated Quantifiler Y Human Male reaction setup was also Quantifiler Y tested across a range of DNA concentrations using both plate and tube configurations Human Male kit 4 source vessels reactions from a plate and tubes Quantifiler Y Figure 25 on page 111 top shows a box and whisker plot of the average DNA Human Male kit concentrations for extracted DNA samples transferred from a 96 well plate Figure 25 results bottom shows the same for samples transferred from microcentrifuge tubes Table 19 on page 113 shows average concentration and standard deviation results In determining the accuracy of automated Quantifiler Y Human Male reaction setup using the HID EVOlution system the greatest standard deviation in the samples at probable range 2 1 ng L varied lt 5 from the average for all three experiments regardless of the source vessel 110 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Precision and reproducibility studies qPCR reaction setup scripts Quantifiler Y Precision Study Plate 100 104 o c D B 1 fo o S Q 0 14 E 0 01 E T T T T T T 0 025 0 05 0 1 1 0 5 0 25 0 DNA Source ng pL Quantifiler Y Precision Study Tube 100 i E aa o c B i
111. iuuejeg E Chapter 6 Prepare STR PCR Reagents and Labware Set up reagents Set up reagents After using the appropriate table see Determine reagent volumes on page 47 to determine the amount of AmpF STR kit reagents and T E buffer that you need to prepare for the run prepare the reagents and place the reagents in the chilled STR PCR reagent block as follows 1 Thaw mix and centrifuge the reagents according to the procedures in the appropriate AmpF STR PCR Amplification Kit User Guide 2 For kits with tubes of AmpliTaq Gold DNA Polymerase To insure correct pipetting by the HID EVOlution system spin down one full 50 uL tube of the AmpliTaq Gold DNA Polymerase then pipette the entire volume into a second full tube of AmpliTaq Gold DNA Polymerase from the same lot for a final volume of 100 uL IMPORTANT The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors or cause the LiHa to crash 4 Prepare the DNA control s as follows 52 If necessary combine reagents from different tubes from the same lot to meet the minimum volume requirements a If necessary to meet your laboratory target concentrations dilute a portion of the control DNA with T E buffer 10 mM Tris HCl pH 8 0 and 0 1 mM Na EDTA to meet the minimum required volume shown in the table No
112. jeg eo epinc peuejs Bue uieisAs dnies H LS HOdb uonniOAd AIH Table 10 MiniFiler and SEfiler Plus kits reagent volumes 25 pL reaction volume for STR PCR amplification Required Extracted Control Required Required Available Volume DNA Excess Dead Vol s Samples Reacti Vol 8 Reagent olume in per amples per Run eactions olume Mns Rearea Vol Full Tube Reaction per Run per Run per Tube inimum Required Volume tor of Reagent 88 Samples and 2 Controls A B C D E AmpF STR Primer Set 500 uL 5 5 uL up to 88 2 4 50 uL A x B C D E 567 uL in one tube AmpF STR Master Mix 500 uL 11 uL up to 88 2 4 50 uL A x B C D E 1084 uLS in one tube AmpF STR Control DNA 007 300 uL 60 uL for 1 to 88 samples plus 2 controls 60 uL T4o9Eo4 buffer in reagent block NA 60 uL for 1 to 88 samples plus 2 controls 60 uL T4o9Eo 4 buffer in trough NA 25 mL in trough for 1 to 88 samples plus 2 controls 25 mL in trough t Includes excess volume to compensate for evaporation and pipetting losses during the run A 50 pL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips If necessary combine reagents from different tubes from the same lot to meet the minimum volume requirements When ordering kits you can request multiple kits from the same lot tt 5 5 pL reaction x 88 2 4 reactions 50 uL tu
113. k Sie a ai Berea Sheet me She Ok See a Eee Ban Sie ac 151 HID EVOlution gPCR STR Setup System Getting Started Guide Purpose Steps covered in this Getting Started Guide HID EVOlution gPCR STR Setup System Getting Started Guide About This Guide This guide provides procedures for human identification HID customers who want to use the HID EVOlution qPCR STR Setup System to automate Reaction plate setup for quantitative real time PCR qPCR DNA normalization and PCR amplification reaction plate setup for Short Tandem Repeat STR analysis Always refer to this guide for pre run and post run handling of samples kit reagents and pipetted PCR plates Refer to Tecan documentation for details on the HID EVOlution qPCR STR Setup System safety set up operating and maintenance instructions Prepare Sample Extract DNA Perform Quantitative PCR Perform routine maintenance Chapter 2 Prepare samples qPCR reagents and system Chapter 3 Run automated qPCR reaction setup Chapter 4 Import a SDS plate record Chapter 5 Run qPCR and review results Chapter 5 Y Perform STR PCR Amplification Perform routine maintenance Chapter 2 Prepare samples STR PCR reagents and system Chapter 6 Run automated DNA normalization and STR PCR reaction setup Chapter 7 Run STR PCR Chapter 8 Steps performed on the HID EVOlution qPCR STR Setup System Perform Capillary Electrophoresis Genetic Analys
114. kit 87 Yfiler kit 88 reagent block cooling 24 46 reagent volumes about 25 47 COfiler kit 51 Identifiler kit 48 MiniFiler kit 50 pre prepared DNA standard dilution series 26 Profiler Plus kit 51 Quantifiler DNA Quantification Kits 26 28 SEfiler Plus kit 50 SGM Plus kit 51 system prepared DNA standard dilution series 27 Yfiler kit 49 reagents combining tubes 52 required amount of DNA 94 required materials qPCR reaction setup 24 STR PCR reaction setup 46 results group creating 11 S safety biological hazards 145 chemical 142 chemical waste 144 guidelines 142 144 sample files create 18 export 7500 Results file 42 import 7500 Setup file 41 import CE Setup file 68 in HID workflow 3 sample information import manually enter and or scan from barcodes 59 options for entering in EVOware software 17 Sample Normalization Adjustment window 59 scripts for maintenance 12 for qPCR reaction setup 34 for STR PCR amplification reaction setup 58 SDS software create detectors 10 export 7500 Results file 42 import the 7500 Setup file 41 153 Index SEfiler Plus kit reagent block configuration 86 required reagent volumes 50 SGM Plus kit reagent block configuration 87 required reagent volumes 51 STR PCR amplification reaction plate layout 62 STR PCR reaction setup maximum samples per run 13 required materials 46 scripts 58 set up extracted DNA in a 96 well plate 14 set up extracted
115. l instructions in steps 3 through 5 below 58 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 7 Run Automated STR PCR Setup T Run STR PCR setup 3 Before entering or importing sample information make sure that the sample IDs that you will import or manually enter match the sample IDs in the 7500 Results file s If you are using barcodes make sure that the barcodes on the extracted DNA samples match the barcodes in the 7500 Results file s 4 After entering sample information either manually by using barcodes or by importing a qPCR STR Samples file a Confirm that the right side of the window displays a message with the number of samples you entered for example 60 60 planned samples present b Ifthe message says that fewer samples will be processed than entered samples for example 715 60 planned samples present this indicates that either Some samples were not given sample information Click Edit to continue entering sample information Too many samples were indicated at the beginning of the wizard Click and start the wizard again this time entering the correct number of samples to process 5 After selecting the appropriate 7500 Results file s view and edit the information in the Sample Normalization Adjustment window a Click View to open the Sample Normalization Adjustment window Note The window lists the number of samples automatically selected for processing and identifies those
116. le ReactionPlatel txt or lt barcode gt txt and saves the file to the C HIDEVOlution qPCRSTR files folder The HID EVOlution system automatically archives the 7500 Setup file in the C HIDEVOlution_qPCRSTRfiles Archive folder when you start the next qPCR or STR PCR setup run If necessary create shortcuts on your desktop to the C HIDEVOlution_qPCRSTRfiles and C HIDEVOlution_qPCRSTRfiles Archive folders P ReactionPlate1 txt Notepad File Edit Format View Help SDS Setup File Version 3 Output Plate Size 96 Output Plate ID ReactionPlatel Number of Detectors 2 Detector Reporter Quencher Description Comments Quantifiler Human FAM IPC VIC well Sample Name Detector Task Quantity 50 000 ng ul Quantifiler Human STND 50 000 Standard 50ng ul IPC UNKN 50 000 ng ul Quantifiler Human STND 50 000 Standard 50ng ul IPC UNKN cfc Quantifiler Human UNKN cfc IPC UNKN 16 700 ng ul Quantifiler Human Standard 16 70ng ul IPC UNKN 16 700 ng ul Quantifiler Human Standard 16 70ng ul IPC UNKN vcag Quantifiler Human UNKN vcgg IPC UNKN 5 560 ng ul Quantifiler Human Standard 5 56ng ul UNKN 5 560 ng ul Quantifiler Human Standard 5 56ng ul UNKN vggtfd Quantifiler Human UNKN vggtfd IPC UNKN 1 850 ng ul Quantifiler Human Standard 1 85ng ul UNKN 1 850 ng ul Quantifiler Human Standard 1 85ng ul UNKN a Quantifiler Human UNKN a IPC UNKN 0 620 ng ul Quantifiler Human Standard 0 62ng ul UNKN 0 620 ng ul Quantifiler Human Sta
117. leeleeeeeeesne 54 Prepare qPCR Reagents and Labware Run Automated qPCR Setup Perform qPCR and Review Results Chapter 6 Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution qPCR STR Setup System Getting Started Guide 45 Chapter 6 Prepare STR PCR Reagents and Labware Review pre run checklist for STR PCR reaction setup Review pre run checklist for STR PCR reaction setup E Cool the STR PCR reagent block to 4 C before use to help keep reagents cool on the worktable It is recommended when not in use that you store reagent blocks in a refrigerator at 4 C O Optional Locate the qPCR STR Sample file the csv sample file that you created and used in qPCR reaction setup or Optional Create a qPCR STR Sample file that you can use to import sample information during the run as described in Create a qPCR STR Sample file on page 18 Confirm that the 7500 Results file s that you want to use are in csv format and in a location where the file s can be imported to the HID EVOlution qPCR STR Setup System software See Analyze and export the qPCR results on page 42 Assemble the materials you will use See Required instruments software and materials on page 6 for a complete list of materials and sources e Extracted DNA samples Use the plate or tubes containi
118. levant precautions For more information For information on e HID EVOlution gPCR STR Setup System Getting Started Guide DNA standard dilution series preparation and quantitation procedures see the appropriate Quantifiler Kits User s Guide DNA normalization and amplification procedures see the appropriate AmpF STR Kit User Guide as noted in Applied Biosystems documentation on page 148 Manual DNA extraction procedures see the PrepFiler Forensic DNA Extraction Kit User Guide Automated DNA extraction procedures see the PrepFiler Automated Forensic DNA Extraction Kit Getting Started Guide Validation experiments performed by Applied Biosystems and the results see Appendix E Validation Experiments and Results on page 103 The HID EVOlution gPCR STR Setup System see the Tecan documentation on page 149 For more information Chapter 1 The HID EVOlution qPCR STR Setup System 8 HID EVOlution qPCR STR Setup System Getting Started Guide The HID EVOlution qPCR STR Setup System Chapter 2 Pre Run Procedures Prepare qPCR Reagents and Labware Run Automated qPCR Setup Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution gPCR STR Setup System Getting Started Guide Chapter 2 Pre Run Procedures This chapter provides prerequisi
119. luding failed samples Correct samples in wells 14 through 61 Incorrect HID EVOlution qPCR STR Setup System Getting Started Guide Set up extracted 1 DNA samples in tubes HID EVOlution gPCR STR Setup System Getting Started Guide Chapter 2 Pre Run Procedures 2 Before each run Set up extracted DNA samples Confirm the number and labeling of tubes For qPCR setup confirm that you have no more than 80 labeled 1 5 mL microcentrifuge tubes containing extracted DNA samples or control samples or no more than 32 tubes for a combined plate For STR PCR setup confirm that you have no more than 88 labeled 1 5 mL microcentrifuge tubes 87 for the Yfiler kit containing extracted DNA samples If you use barcodes to track samples confirm that barcodes are correctly placed on the tubes see barcode information in the Tecan HID EVOlution qPCR STR Setup System Application Manual Confirm that the tube racks are correctly positioned For qPCR setup confirm that the tube racks S1 through S5 are at grid positions 27 31 For STR PCR setup confirm that the tube racks S1 through S6 are at grid positions 27 32 Correctly position the sample tubes in the tube racks a Place the first sample tube in the tube racks for example you can begin with rack S1 position 8 b After the first sample tube continue placing sample tubes from back to front in vertical columns as shown in the examples below Do not l
120. m EVO instrument refer to the manufacturer s instrument documentation 1 54 Set up the DiTis as described in the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 4 3 5 Set Up Plasticware and Samples on the Workstation Remove caps from the reagents then place the loaded reagent block Figure 5 item 8 on grid 15 site position 1 See Tables 8 through 11 on pages 48 through 51 for correct volumes Place T E buffer for dilution of DNA samples into a 100 mL trough Figure 5 item 7 then place the trough on grid 14 site position 2 See Tables 8 through 11 on pages 48 through 51 for correct volumes Place three empty MicroAmp Optical 96 Well Reaction Plates into the metal plate adapter with well A1 in the top left corner Figure 5 items 10 12 on Grid 15 site position 2 pre dilution plate Grid 21 site position 2 pre dilution plate e Grid 21 site position 3 STR PCR reaction plate Reaction 2 3 4 5 8 7 8 9 w um Reaction plate plate well 1 notch A1 IMPORTANT To ensure that samples are transferred to the correct wells confirm for each plate that The plate is placed in the metal plate adapter The plate wells are aligned with the holes in the metal plate adapter e Well AI is positioned in the upper left corner HID EVOlution qPCR STR Setup System Getting Started Guide Set up the labware on the worktable Chapter 6 Prepare STR PC
121. mL tube s of Quantifiler Human Primer Mix Place the first tube in position 9 If you use more than one tube continue to position 10 then position 11 12 Quantifiler PCR Reaction mix 13 Empty 5 mL VWR tube for the master mix 14 Quantifiler Human DNA Standard tube t The required number of tubes with the Quantifiler Human Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 pL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 pL plus a 50 pL dead volume If the volume of reagent in the first tube position 9 falls below 240 uL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid HID EVOlution qPCR STR Setup System Getting Started Guide 7 Appendix B Reagent Block Configurations Quantifiler DNA Quantification Kits Quantifiler Y Human Male Kit with pre prepared standards Figure 10 Reagent block configuration for Quantifiler Y Human Male kit with pre prepared DNA standards Legend 1 8 1 5 mL tubes of pre prepared DNA standards arrange concentrations as shown in the corresponding orange table 9 11 Up to three 1 5 mL tube s of Quantifiler Y Human Male Primer Mix Place the first tu
122. me Any higher dilutions are performed as two step dilutions The HID EVOlution system will dilute a sample with a greater concentration than the target quantity maximum transferable volume at least once 1 The system performs the first dilution D1 Note The maximum dilution ratio for D1 is 1 20 for a 25 uL total reaction volume and 1 40 for 50 uL total reaction volumes 2 If the first dilution D1 is sufficient to reach the target amount of DNA the HID EVOlution system transfers the maximum allowable volume of sample either 10 or 20 uL from DI to the STR PCR reaction plate HID EVOlution gPCR STR Setup System Getting Started Guide Appendix D Dilution Protocols About DNA normalization on the HID EVOlution system 3 Ifthe first dilution D1 is still too concentrated to reach the target amount of DNA then the HID EVOlution system performs a second dilution D2 Note The maximum dilution ratio for D2 is 1 20 for a 25 uL total reaction volume and 1 40 for 50 uL total reaction volumes 4 The HID EVOlution system transfers the maximum allowable volume of sample either 10 or 20 uL from D2 to the STR PCR plate In a two step dilution the final dilution ratio equals the first dilution times the second dilutions For example two dilutions each of 1 20 will result in a 1 400 final dilution ratio The workflows in Figure 21 are for demonstration purposes only Workflow for normalization using a 10 pL addit
123. mple used in a 1 or 2 step dilution is not included in the STR Samples Report provided at the end of the run To determine the amount 1 Determine the dilution ratio used for the sample The final dilution ratio for each sample is shown in the Sample Normalization Adjustment window The STR Samples report also shows the dilution ratio s If you are using the information from the Samples report to determine the dilution ratio for a 2 step dilution you will need to multiply the ratio for each of the steps together to determine the final dilution ratio 2 Locate the dilution ratio in Table 15 on page 100 for a 10 uL addition or in Table 16 on page 101 20 uL addition The volume of extracted DNA sample used is shown in the same row in the next column This is true for any target DNA input amount but note that the first 2 columns of these tables apply only for 1 ng target DNA input amounts HID EVOlution qPCR STR Setup System Getting Started Guide Appendix D Dilution Protocols Dilution protocols with examples Dilution protocol Example of no dilution direct transfer If you select a sample for processing that examples has a T Amt target amount less than the Global Req Amount as defined in the Sample Normalization Adjustment window the HID EVOlution system transfers the maximum allowable volume of the sample either 10 or 20 uL to the STR PCR plate For example if the Global Req Amount is 1 ng and you select a 0 025 ng uL
124. n BF TE3 TE3 100 Sample None HID_Advar MiniF iler TE3 HIDEvolution HIDEvolution 12 G1 5O0ng4 5O0ng4 100 Sample None HID_Advar MiniFiler 5 ng4 HIDEvolution HIDEvolution 13 H1 TE4 TE4 100 Sample None HID_Advar MiniF iler TE4 HIDEvolution HIDEvolution 14 A2 TES TES 100 Sample None HID Advar MiniFiler TES HiDEvolution HiDEvolution 15 B2 5 ng5 5 ng5 100 Sample None HID_Advar MiniF iler 50ng5 HIDEvolution HIDEvolution 16 C2 TEG TEG 100 Sample None HID_Advar MiniFiler TE6 HIDEvolution HIDEvolution 17 D2 10ng1 10ng1 100 Sample None HID_Advar MiniFiler 10ng1 HIDEvolution HIDEvolution 18 E2 TE TE 100 Sample None HID_Advar MiniFiler TE HIDEvolution HIDEvolution 19 F2 Positive Positive 100 Positive CcNone HID Advar MiniFiler Positive HIDEvolution HiDEvolution 20 G2 Negative Negative 100 Negative C None HID_Advar MiniFiler Negative HIDEvolution HIDEvolution 211H2 ladder ladder 100 Allelic Lad None HID Advar MiniF iler ladder HiDEvolution HiDEvolution HID EVOlution qPCR STR Setup System Getting Started Guide Note By default the instrument protocol and the results group are both named HIDEvolution however individual systems can have different configurations Confirm that in the Data Collection software you have created an instrument protocol and a results group with the same names that you noted in step 2 If not follow steps 4 and 5 to create them If necessary set up th
125. n the SDS software click Analyze b Omit any blank samples c Review the standard curve to ensure that results are within the recommended range see the Quantifiler Kits User s Manual If necessary omit standard outliers then click Analyze to recalculate the standard curve d Review the quantitation data including the IPC Cy and consider the condition of the original sample to determine if any samples require additional processing before STR PCR reaction setup 2 When you are done analyzing the results in the SDS software export the 7500 Results file and copy to the HID EVOlution system computer a In the SDS software select File Export Results b Navigate to the desired location on your hard drive c Type a name for the exported file d Select the export type csv 42 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 5 Perform qPCR and Review Results 5 For more information e Save the file f Copy the file to the computer where the EVOware Software is installed or to a server that can be accessed from both systems Note During STR PCR reaction setup you import the sample information in the 7500 Results file to the HID EVOlution qPCR STR Setup System The EVOware uses the information in the 7500 Results file to set up sample information and to help normalize the DNA input amount for STR PCR reaction setup on the HID EVOlution gPCR STR Setup System For more informa
126. ndard 0 62ng ul UNKN 0 210 ng ul Quanti fil Human Standard 0 21ng ul UNKN 0 210 ng ul Quanti fil Human Standard 0 21ng ul UNKN 0 068 ng ul Quanti fil Human Standard 0 068ng ul UNKN 0 068 ng ul Quanti Fi Human Standard 0 068ng ul NKN 0 023 ng ul Quanti fil Standard 0 023ng ul 0 023 ng ul Quanti fi Standard 0 023ng ul 40 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 5 Perform qPCR and Review Results S Transfer and import the 7500 Setup file Transfer and import the 7500 Setup file IMPORTANT To successfully import the 7500 Setup file to the SDS software the detector names created in the SDS software must match those in the 7500 Setup file See Create detectors for the 7500 Real Time PCR System on page 10 1 Transfer the 7500 Setup file generated by the HID EVOlution system at the end of the qPCR reaction setup run to the SDS software computer a On the HID EVOlution system navigate to the C HIDEVOlution_qPCRSTRfiles folder then locate the file ReactionPlate1 txt or lt barcode gt txt b Optional Rename the file c Copy and transfer the ReactionPlatel txt or lt barcode gt txt file to a location where it can be accessed by the SDS software computer 2 Start the 7500 Real Time PCR System and SDS Software a Start the computer b Power on the 7500 instrument c Start the SDS Software Note For more information on starting the 7500 Real Time PCR
127. ndard dilution series preparation and reaction setup Experiment Results To evaluate the ability of the HID EVOlution system to precisely prepare and transfer a DNA standard dilution series 40 DNA standard dilution series were prepared on the system The plates were prepared using Quantifiler Human and Quantifiler Y Human Male DNA Quantification kits The DNA standard dilution series consisted of eight concentrations 50 ng uL 16 7 ng uL 5 56 ng L 1 85 ng uL 0 62 ng uL 0 21 ng uL 0 068 ng uL and 0 023 ng uL Two replicates of each concentration from each of the 40 DNA standard dilution series were transferred by the system to a plate creating a total of eight plates Each plate also contained a manually prepared DNA standard dilution series The eight plates were then run on a single 7500 Real Time PCR System For qPCR the standard thermal cycling protocol described in the Quantifiler Kits User s Manual Chapter 3 PN 4344790 was used for all 7500 instrument runs To evaluate the reproducibility and precision of the automated DNA standard dilution series setup across multiple 7500 instruments an additional 35 DNA standard dilution series were prepared by the HID EVOlution system and run on three different 7500 instruments for a total of 75 DNA standard dilution series tested After qPCR the R and slope values calculated by the 7500 System SDS Software v1 2 3 were compared to the original Quantifiler kit validation re
128. netic Analyzer Data Collection Software version 3 0 Applied Biosystems Contact your local sales representative for computer configurations Table 3 Required materials Material Sourcet Benchtop centrifuge with 96 well plate adapters Disposable pipette tips DiTis 50 yL LiHa conductive disposable tips with filter Tecan PN 30032114 www tecan com Disposable pipette tips DiTis 200 yL LiHa conductive disposable tips with filter Tecan PN 30000629 www tecan com 100 mL disposable troughs for reagents Tecan PN 10613048 www tecan com Barcodes optional See the Tecan HID EVOlution gPCR STR Setup System Application Manual and the Tecan Freedom EVO Operating Manual Section 3 5 6 Positive Identification PosID for requirements MicroAmp clear adhesive film to seal PCR product plates for storage Applied Biosystems PN 4306311 MicroAmp adhesive film applicator Applied Biosystems PN 4333183 MicroAmp optical adhesive film to seal qPCR reaction plates during qPCR Applied Biosystems PN 4360954 Four 96 well reaction plate adapters supplied with the system listed as adapter plate PCR HID EVOlution in the HID EVOlution qPCR STR Setup System Application Manual Note If you are processing extracted DNA from plates you will require 2 adapters for qPCR setup and 4 adapters for STR PCR setup If you are processing extracted DNA f
129. ng extracted DNA samples that you prepared and processed in the associated qPCR reaction setup run See Set up extracted DNA samples in a 96 well plate on page 14 or Set up extracted DNA samples in tubes on page 15 e Reagents Applied Biosystems AmpF STR kit Ty Ep buffer 10 mM Tris HCl pH 8 0 and 0 1 mM Na EDTA Refer to your specific AmpFSTR Kit User Guide for directions on buffer preparation Degassed deionized water system liquid 3000 mL per run e Plasticware A MicroAmp Optical 96 Well Reaction Plate to be used as the STR PCR reaction plate Two additional MicroAmp Optical 96 Well Reaction Plates to be used as pre dilution plates One tray of 200 uL DiTis for 96 reactions Three trays of 50 uL DiTis for 96 reactions e HID EVOlution gPCR STR Setup System carriers Four 96 well metal microplate plate adapters Up to six 16 position tube carriers with vertical cap storage if extracted DNA samples are in tubes Optional Barcodes for DNA sample plate or tubes and STR PCR plate Make a list of the lot numbers and expiration dates of the AmpF STR kit components that will be used in the run for entry into the HID EVOlution qPCR STR Setup System software Set up the carriers and racks according to the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 4 3 3 Set Up Carriers and Racks OOO Start up the system and perform routine maintenanc
130. ntifiler PCR Amplification Kit 4322288 AmpF STR MiniFiler PCR Amplification Kit 4373872 AmpF STR Profiler Plus PCR Amplification Kit 4303326 AmpF STR SEfiler Plus PCR Amplification Kit 4382699 AmpF STR SGM Plus PCR Amplification Kit 4307133 AmpF STR Yfiler PCR Amplification Kit 4359513 Refer to the appropriate Quantifiler kit and AmpF STR kit user guides for kit contents and storage conditions HID EVOlution qPCR STR Setup System Getting Started Guide 5 Required instruments software and materials Chapter 1 The HID EVOlution qPCR STR Setup System Required instruments software and materials The additional instruments software and materials needed for use but not supplied with the HID EVOlution qPCR STR Setup System are described in Tables 2 and 3 Table 2 Required instruments and software Instrument or Software Source Applied Biosystems 7500 Real Time PCR System and Sequence Detection Software v1 2 3 Applied Biosystems Contact your local sales representative for computer configurations 9600 9700 Gold plated Silver 96 Well GeneAmp PCR System or 9600 9700 Silver 96 Well GeneAmp PCR System or Veriti 96 well thermal cycler with silver 96 well sample block or gold plated silver 96 well sample block Applied Biosystems PN 4314878 Applied Biosystems PN N8050001 Applied Biosystems PN 4375786 i Applied Biosystems 3130 3130xl Genetic Analyzer and Ge
131. ntrifuge according to the Quantifiler Kits User s Manual 2 If you are using a previously prepared DNA standard dilution series vortex and briefly centrifuge the DNA standard dilutions at low speed before use 3 Select the appropriate reagent block setup from the following table then use the appropriate figure to place the reagents and labeled empty tubes in the reagent block IMPORTANT If you are using a pre prepared DNA standard dilution series make sure to place each concentration in the correct location in the reagent block Set up the If you are using And the DNA standards reagent block according to The Quantifiler Are pre prepared Figure 8 on Human kit page 76 Will be prepared by the HID EVOlution Figure 9 on system page 77 The Quantifiler Y Are pre prepared Figure 10 on Human Male kit page 78 Will be prepared by the HID EVOlution Figure 11 on system page 79 Both the Are pre prepared Figure 12 on Quantifiler page 80 ee ee Will be prepared by the HID EVOlution Figure 13 on system page 81 HID EVOlution gPCR STR Setup System Getting Started Guide 29 Chapter 3 Prepare qPCR Reagents and Labware Set up the labware on the worktable Set up the labware on the worktable 30 After loading the reagent block use the following procedure to place the reagent block PCR reaction plate trough disposable pipette tips DiTis and samples on the worktable Use Figure
132. nually set up control DNA samples using the same reagents and instrumentation to confirm that the reagents protocol equipment and instrumentation are functioning as expected Use the maximum volume permitted for the DNA sample If possible repurify DNA and requantify Otherwise use the minimal volume possible Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide Verify that correct TE buffer was used with 0 1 mM EDTA Before the next run To ensure correct pipetting clean and finger tighten the DiTi cones diluter valves and syringe fittings Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual Confirm that you have sufficient system liquid then flush the system and check for air bubbles and leaks Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual HID EVOlution qPCR STR Setup System Getting Started Guide 73 Appendix A Troubleshooting 74 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix B Reagent Block Configurations This appendix provides the reagent block configurations referred to in Chapter 3 Prepare qPCR Reagents and Labware on page 23 and Chapter 6 Prepare STR PCR Reagents and Labware on page 45 for the following kits IMPORTANT Prepare the reagents as directed in Set up reagents on page 25 and Set up reagents on page 52 before pla
133. o not use Microsoft Excel which may introduce invalid formatting b Select File Open then browse to the template files originally provided in the Sample Files folder on the HID EVOlution qPCR STR Setup System software CD c Select SampleFile 96 csv if using tubes or SampleFile Plate 96 csv if using a plate then click Open 2 Select File Save As browse to the directory C HIDEVOlution_qPCRSTRfiles or another location of your choosing change the file name to lt UserDefined gt csv then click Save HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 2 Pre Run Procedures 2 Before each run Optional Set up sample information 3 When editing a template follow the formatting rules that are described in Tecan HID EVOlution gPCR STR Setup System Application Manual Section 3 4 2 File Format for Plate Wells Tubes and the following guidelines Do not include empty plate well or tube rack positions between samples Avoid spaces or other special characters such as commas asterisks or slashes Forthe sample name field follow your laboratory naming conventions to assign a unique name to each sample Make sure that the sample name meets the formatting rules 4 Save the file with a csv extension then close the file IMPORTANT The file extension must be csv for the file to be imported to the HID EVOlution software HID EVOlution gPCR STR Se
134. o obtain support on page x HID EVOlution gPCR STR Setup System Getting Started Guide Numerics 1 5 mL tubes for extracted DNA samples 15 3130 3130x Genetic Analyzer results group and instrument protocol 11 7500 Real Time PCR System detectors 10 7500 Results file export from SDS software 42 in HID workflow 3 location of file 42 7500 Setup file import to SDS software 41 in HID workflow 3 location of file 40 96 well plate for extracted DNA samples 14 A AmpF STR PCR Amplification Kits reagent block configurations 82 required reagent volumes 48 49 50 51 supported for use with HID EVOlution qPCR STR Setup System 5 AmpliTaq Gold DNA Polymerase preparing 52 Applied Biosystems customer feedback on documentation 150 Information Development department 150 automation on other liquid handling platforms 135 qPCR scripts 34 STR PCR scripts 58 B barcodes scan for sample information 17 59 biohazardous waste handling 145 C calculate reagent volumes AmpF STR kits 47 Quantifiler DNA Quantification Kits 26 CAUTION description ix CE Setup file import for CE analysis 68 in HID workflow 3 location of file 67 prerequisites for importing 11 HID EVOlution gPCR STR Setup System Getting Started Guide Index CE signal troubleshooting high 72 troubleshooting low 73 chemical safety 142 chemical waste safety 144 COfiler kit reagent block configuration 82 required reagent volumes 51 combin
135. of pre prepared DNA standard dilution series arrange concentrations as shown in the corresponding orange table 9 11 Up to three 1 5 mL tube s of Quantifiler Human Primer Mix Place the first tube in position 9 If you use more than one tube continue to position 10 then position 11 12 Quantifiler PCR Reaction mix 13 Empty 5 mL VWR tube for the master mix t The required number of tubes with the Quantifiler Human Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 uL plus a 50 pL dead volume If the volume of reagent in the first tube position 9 falls below 240 uL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid 76 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix B Reagent Block Configurations E Quantifiler DNA Quantification Kits Quantifiler Human Kit with system prepared standards m n OP Figure 9 Reagent block configuration for Quantifiler Human kit with DNA standards prepared by the HID EVOlution system Legend 1 8 1 5 mL empty tubes for DNA standard dilution series 9 11 Up to three 1 5
136. ompare the results to those for samples set up manually 0 025ng ul 0 05ng ul 0 1ng ul 1ng ul 5ng ul 25ng ul Figure 24 Quantification results comparing Quantifiler Human kit reactions set up manually and with the HID EVOlution system n 212 109 E Appendix E Validation Experiments and Results Precision and reproducibility studies qPCR reaction setup scripts Table 18 Average quantity and standard deviation for quantification of human DNA using the Quantifiler Human DNA Quantification kit Sample Mic Tube1 Tube2 Tube3 Platei Plate2 Plate3 Manual Average quantity ng pL 25 12 29 458 33 984 30 809 25 043 28 388 25 512 29 654 5 12 5 743 6 116 5 435 4 646 5 037 5 052 5 930 1 12 1 140 1 129 1 107 0 927 0 932 1 072 1 161 0 1 12 0 132 0 136 0 114 0 090 0 091 0 120 0 127 0 05 12 0 067 0 065 0 070 0 050 0 047 0 066 0 073 0 025 12 0 033 0 040 0 032 0 031 0 030 0 050 0 039 Standard deviation 25 12 0 793 1 342 0 701 2 538 1 336 1 238 1 488 5 12 0 177 0 232 0 198 0 201 0 132 0 146 0 330 1 12 0 075 0 057 0 037 0 050 0 054 0 062 0 063 0 1 12 0 016 0 015 0 022 0 011 0 012 0 021 0 014 0 05 12 0 015 0 013 0 010 0 009 0 010 0 011 0 018 0 025 12 0 010 0 009 0 006 0 010 0 011 0 018 0 016 Quantifiler Y Human Male kit precision study Experiment The experiment described in Quantifiler Human reaction setup precision and reproducibil
137. on qPCR STR Setup System Detailed Workflow Description zuerst Gre DR REQUE BR CAE wer e t oca e de de andes 89 Dilution Protocols oxen thea Suy et ae led rep ees RD Mer ut 93 DNA input amount must be normalized before STR PCR amplification 94 Terms used in the Freedom EVOware software 00 0 cece cece ene eee 94 Guidelines for successful normalization 00 ccc eee 94 About DNA normalization on the HID EVOlution system 000 cece eee eee 96 Dilution protocols with examples 00 00 cee 98 Validation Experiments and Results 000000 eee 103 OVerVvieW sy nini giroen mod ima Mba ER OE ER aati odisea ae ERU disease 104 Materials and methods 0 0000 cee hrs 105 Precision and reproducibility studies DNA standard dilution series preparation and reaction setup 0000 cee eere 107 Precision and reproducibility studies qPCR reaction setup scripts 108 Quantifiler Human reaction setup precision and reproducibility study 108 Quantifiler Y Human Male kit precision study 000 cece cece eee ee eee 110 Combined Quantifiler Human and Quantifiler Y Human Male reaction setup precision Study seren 2084 meret Rie eon ene e Ruhe ean ded RR Ren ee Xen na 111 Precision and reproducibility studies STR PCR amplification reaction setup scripts 114 Identifiler and SGM Plus STR PCR amplification reaction setup precision and reproducibilit
138. on Kit Getting Started Guide Perform Quantitative PCR 1 Perform routine maintenance 2 Pre Run Procedures on page 9 2 Prepare samples qPCR reagents and system 3 Prepare qPCR Reagents and Labware on page 23 3 Run automated qPCR reaction setup 4 Run Automated qPCR Setup on page 33 4 Import a SDS plate record 5 Perform qPCR and Review Results on page 39 5 Run qPCR and review results 5 Perform qPCR and Review Results on page 39 Perform STR PCR Amplification 1 Perform routine maintenance 2 Pre Run Procedures on page 9 2 Prepare samples STR PCR reagents and system 6 Prepare STR PCR Reagents and Labware on page 45 3 Run automated DNA normalization and STR PCR 7 Run Automated STR PCR Setup on page 57 reaction setup 4 Run STR PCR amplification 8 Perform STR PCR and Set Up Capillary Electrophoresis on page 65 Perform Capillary Electrophoresis Genetic Analysis 1 Import a CE plate record 8 Perform STR PCR and Set Up Capillary Electrophoresis on page 65 2 Set up CE reactions 3 Run CE CE and data analysis procedures are not included in this han guide See the AmpF STR Kit User s Guides Perform Data Analysis HID EVOlution gPCR STR Setup System Getting Started Guide Chapter 1 The HID EVOlution qPCR STR Setup System 1 HID EVOlution qPCR STR Setup System workflow Sample Instrument Software Sample Information Informat
139. on reaction setup Place extracted DNA samples in 96 well plates or 1 5 mL tubes Prepare AmpF STR kit reagents for use on the HID EVOlution qPCR STR Setup System Prepare the HID EVOlution gGPCR STR Setup System b Run automated STR PCR reaction setup on the HID EVOlution qPCR STR Setup System The HID EVOlution system Reads the sample information from either the imported qPCR STR Sample input file csv or the manually entered sample list Reads the 7500 Results file s that you import from the 7500 Real Time System SDS Software v1 2 3 Confirms that the sample ID names from the Sample input file or sample list match the 7500 Results output file s Determines the dilution ratio for each sample if required based on the measured DNA concentration the required DNA input volume or the user defined target DNA input amount for the STR PCR reactions Dilutes the extracted DNA samples as needed to normalize the sample concentrations Prepares the master mix Transfers the master mix and normalized DNA samples into a STR PCR reaction plate and mixes the reaction components Generates a CE Setup file that can be imported into to the CE instrument 3130 3130x Genetic Analyzer Data Collection Software v3 0 to automatically populate the plate record Note The HID EVOlution gPCR STR Setup System can prepare up to 88 STR PCR reactions 87 when using the AmpF STR Yfiler PCR Amplification
140. ors 82 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix B Reagent Block Configurations AmpF amp TR PCR Amplification Kits AmpF STR Identifiler PCR Amplification Kit Figure 15 Reagent block configuration for Identifiler kit Legend 1 Control DNA 9947A diluted as necessary See step 4 on page 52 2 1 5 mL tube containing T o5E buffer 3 Empty VWR tube for master mix preparation 4a 4b AmpF STR PCR Reaction Mix 5 Identifiler Primer Mix 6 AmpliTag Gold DNA polymerase t You must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors HID EVOlution qPCR STR Setup System Getting Started Guide 83 Appendix B Reagent Block Configurations AmpF amp STR PCR Amplification Kits AmpF STR MiniFiler PCR Amplification Kit Figure 16 Reagent block configuration for MiniFiler kit Legend 1 Control DNA 007 diluted as necessary See step 4 on page 52 2 1 5 mL tube containing Tj E94 buffer 3 Empty VWR tube for master mix preparation 4a 4b MiniFiler Master Mix 5 MiniFiler Primer Mix 84 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix B Reagent Block Configurations AmpF amp TR PCR Amplification Kits AmpF STR Profiler Plus PCR Am
141. pipetting that the reagents protocol equipment and occurred because of instrumentation are functioning as expected Incorrect or improperly e Confirm all necessary reagents are present and placed DiTis plates tubes correctly positioned on the workstation Refer to or hardware the Tecan HID EVOlution qPCR STR Setup Air bubbles or leaks in System Application Manual system e Confirm that you use the specified DiTis plates tubes and metal racks and carriers in the correct Diny orloose DiTicones positions Refer to the Tecan HID EVOlution DiTis vel not picked up qPCR STR Setup System Application Manual propery e Clean and finger tighten the DiTi cones diluter Reagent equipment or valves and syringe fittings Refer to the Tecan instrumentation failure HID EVOlution qPCR STR Setup System Application Manual e Confirm that you have sufficient system liquid then flush the system and check for air bubbles and leaks Refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual Review the LiHa coordinates x y and z positions of the DiTis For details contact Applied Biosystems or refer to the Tecan Freedom EVOware Standard 2 1 Freedom EVOware Plus 2 1 Extended Device Support Software Manual Section 9 4 4 Teaching the Labware Coordinates e Amplify the maximum volume for STR analysis e Extract DNA from a different sample that is prepared from the same source e
142. plays the error message Not enough liquid HID EVOlution gPCR STR Setup System Getting Started Guide 25 3 Chapter 3 Prepare qPCR Reagents and Labware Set up reagents Determine required Use the following procedure to determine the amount of Quantifiler kit reagents and reagent volumes T E buffer that you need to prepare for the run Note Applied Biosystems recommends that you combine reagents from different tubes from the same lot if necessary to meet the minimum volume requirements If you want to Usea DNA standard dilution series that was prepared in the last two weeks Start with step 1 below Let the HID EVOlution system prepare the DNA standard dilution series for you Start with step 2 on page 27 1 Optional If you want to use a pre prepared DNA standard dilution series determine if the series meets the following requirements The series must have been prepared in the last two weeks The series must have been prepared according to the procedures specified in the appropriate Quantifiler Kits User s Manual You must have the required volumes of the pre prepared DNA standard dilutions shown in the table Required Volumes for Pre Prepared DNA Standard Dilution Series Type of qPCR reaction plate Required Volume Plate prepared with EITHER Quantifiler Human or Quantifiler Y Human Male Quantification Kit 60 uL of each of the eight concentrations of diluted standard Note Thi
143. ple to sample reproducibility was observed among all ten replicates in each of the eight sample concentrations A maximum standard deviation was observed at the 50 ng uL concentration of 1 104 Other standard deviations were lt 0 2 indicating minimal sample to sample variation DNA Quantification zr z Z a ANA 66069 GO ONANAAAD SP 9 9 9 9 9999 SSS 1 Source DNA ng pL Figure 40 Quantitative results from complete system check study Figure 41 on page 129 shows the results of the STR PCR amplification analysis Extracted DNA samples were first quantified using the Quantifiler Human script then normalized for the Identifiler PCR reaction setup n 176 The figure shows the average allele peak height for all the replicates normalized across a range of DNA concentrations The peak height averages were within the acceptable range of 1000 3000 RFU 128 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results E Complete system check and precision study Identifiler Complete System Study 40004 2 30004 9 7 2 2000 4 9 Oo S S 2 10004 0 1 T T T T T T T T T ly Ln e QO e Q o aS e e SE S 8 Source DNA ng uL a Figure 41 Identifiler kit complete system study The following figures show the Identifiler kit intralocus balance and intracolor balance profiles The profiles were evaluated as a measu
144. plification Kit Figure 17 Reagent block configuration for Profiler Plus kit Legend 1 Control DNA 9947A diluted as necessary See step 4 on page 52 2 1 5 mL tube containing Tj Ep buffer 3 Empty VWR tube for master mix preparation 4a 4b AmpF STR PCR Reaction Mix 5 Profiler Plus Primer Mix 6 AmpliTag Gold DNA polymerase t You must combine the volumes in two full 50 uL tubes in one tube The tube containing the combined volume must be one of the original AmpliTaq Gold DNA Polymerase tubes provided with the kit Use of another type of tube can cause liquid detection errors HID EVOlution qPCR STR Setup System Getting Started Guide 85 Appendix B Reagent Block Configurations AmpF amp STR PCR Amplification Kits AmpF STR SEfiler Plus PCR Amplification Kit D I i Figure 18 Reagent block configuration for SEfiler Plus kit Legend 1 Control DNA 007 diluted as necessary See step 4 on page 52 2 1 5 mL tube containing T o5E buffer 3 Empty VWR tube for master mix preparation 4a 4b SEfiler Plus Master Mix 5 SEfiler Plus Primer Mix 86 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix B Reagent Block Configurations AmpF amp TR PCR Amplification Kits AmpF STR SGM Plus PCR Amplification Kit Figure 19 Reagent block configuration for SGM Plus kit Legend 1 Control DNA 007 diluted as necessary See step 4 on page 52
145. r Set and PCR Reaction Mix or Master Mix the volume required per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For these reagents the instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL this is 190 uL plus a 50 uL dead volume When the reagent volume falls below 240 uL the instrument is likely to determine that the liquid level is too low for successful aspiration If the instrument determines that the liquid level is too low the instrument then looks for a sufficient volume in the next available tube of the same reagent if available or pauses the run and displays the error message Not enough liquid e For the T 4E buffer in the trough the volume necessary to ensure adequate buffer for any dilution ratio required for sample normalization Reagent volume Use the appropriate table below to determine the required volumes of reagents For the tabl m abiss Identifiler kit use Table 8 on page 48 e Yfiler kit use Table 9 on page 49 MiniFiler and SEfiler Plus kits use Table 10 on page 50 COfiler Profiler Plus and SGM Plus kits use Table 11 on page 51 Then prepare the reagents according to Set up reagents on page 52 HID EVOlution gPCR STR Setup System Getting Started Guide 47 oo epinc peuejs Bue uieisAg dnies HLS HOdb uonniOAd AIH
146. re 3 Set up the labware on the worktable 4 Place an empty MicroAmp Optical 96 Well Reaction Plate for the qPCR reactions into the metal plate adapter with well A1 in the top left corner on grid 21 site position 1 see Figure 2 item 9 Reaction 2 3 4 5 8 7 8 9 1 Reaction plate fu S plate weli notch id S zo 2e 94 2 eo eo 00 ve en o Lo o opor os eo s 50 er 75 m wi e 2 20 20 20 44 62 o o ze f o LS 13 24 20 37 46 9 1 09 77 e e 22 o se ae se e2 o ro 0s o osos 2 oo 7 ss eo n o o 4 Lo 16 24 92 oo oo oo 04 72 oo oo IMPORTANT To ensure that samples are transferred to the correct wells confirm that The reaction plate is placed in the metal plate adapter The reaction plate wells are aligned with the holes in the metal plate adapter e Well AI is positioned in the upper left corner 5 Ensure that the extracted DNA is in either a 96 well plate or in 1 5 mL tubes before setting up the samples on the worktable for qPCR setup For instructions see Set up extracted DNA samples in a 96 well plate on page 14 or Set up extracted DNA samples in tubes on page 15 6 Ifthe extracted DNA is in a plate place the extracted DNA sample plate into the metal plate adapter on the worktable in grid 21 site position 3 with well A1 in the top left corner Reaction Reaction pate 2 3 4 5 68 7 8 9
147. re of the relative changes across the loci and the dye sets The intralocus balance for almost all samples containing 2 0 1 ng uL DNA concentrations was gt 70 Figure 42 on page 130 and within the acceptable threshold The intracolor balance was gt 40 Figure 43 on page 130 HID EVOlution qPCR STR Setup System Getting Started Guide 129 E Appendix E Validation Experiments and Results Complete system check and precision study Identifiler Complete System Integration Study Intra locus Balance Marker aua j j E ame E csriPo lg 51 25 80 0195433 E E X B 021511 E 0251338 60 m Bl 0351358 hi BB 55216 Bl 575220 m 40 B 0851179 FGA E rox vwa 20 0 AY Source DNA ng ul Figure 42 Intralocus balance for Identifiler kit complete system study cn ae T plis j Balance 96 EE mn 20 o samples ng u RRITE RIRA PARLOR RRAS Dyes e 4 d QR e Figure 43 Intracolor balance for Identifiler kit complete system study 130 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results Concordance and position ID confirmation study Concordance and position ID confirmation study Experiment For simplicity and consistency during qPCR and STR analyses the controls and allelic ladder positions on a PCR plate are predetermined As a result sample positions in the source and PCR plates may be inconsistent Sample posi
148. rget DNA input amount for each sample Click OK to exit the Sample Normalization Adjustment window Note The software shows a summary of the total number of samples that will be processed HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 7 Run Automated STR PCR Setup Perform post run tasks Perform post run tasks Take care of the 1 Remove the prepared STR PCR reaction plate containing the STR PCR reactions STR PCR reaction from the worktable plate 2 Seal the STR PCR reaction plate with MicroAmp Clear Adhesive Film PN 4306311 3 Place the STR PCR reaction plate in a table top centrifuge with plate holders then centrifuge the plate at 3000 rpm for approximately 20 seconds to remove any air bubbles Clean up the 1 Remove the TE buffer trough and dispose of any remaining TE buffer If it is the instrument last run of the day dispose of the TE buffer trough IMPORTANT Do not reuse the reagents in the troughs See Waste disposal on page 144 2 Follow the instructions in the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 1 After Run on page 108 HID EVOlution qPCR STR Setup System Getting Started Guide 61 Chapter 7 Run Automated STR PCR Setup About the STR PCR plate layout About the STR PCR plate layout Regardless of the extracted DNA sample setup the STR PCR reaction plate is always set up in the same way the sample from the fi
149. rol DNA needs to be diluted dilute a portion of the control DNA with low TE buffer and place the diluted control tube into the reagent rack for amplification setup Ensure that the final volume of the diluted control DNA is at least 50 uL Calculate the volume of each component needed to prepare the reactions using the reactions table below Note Include at least 1096 excess reactions in the calculations to compensate for the loss that occurs during reagent transfers For MiniFiler and SEfiler Plus kits add the required volume of Master Mix and Primer Mix only No additional polymerase is needed Target concentrations and input volumes Volume Per Reaction pL Component a seme TM Profiler SEfiler SGM COfiler Identifiler MiniFiler Plus Plus Plus Yfiler Primer Mix 11 0 5 5 5 0 11 0 5 0 11 0 5 0 PCR Reaction Mix 21 0 10 5 NA 21 0 NA 21 0 9 2 AmpliTag Gold 1 0 0 5 NA 1 0 NA 1 0 0 8 Polymerase MiniFiler or SEfiler NA NA 10 0 NA 10 0 NA NA Plus Master Mix 138 To prepare the reactions 1 Dispense the required volume of primer mix into a separate empty tube for Master Mix preparation 2 Dispense the required volume of PCR reaction mix into the Master Mix tube then mix well 3 Dispense the required volume of AmpliTaq Gold Polymerase into the Master Mix tube then mix well HID EVOlution gPCR STR Setup System Getting Started Guide Appendix F
150. rom tubes you need 1 adapter for qPCR setup and 3 for STR PCR setup Tecan PN 30032860 Six tube racks optionally supplied with the system also listed as 16 position tube carrier with vertical cap storage in the HID EVOlution qPCR STR Setup System Application Manual Tecan PN 10613035 HID EVOlution qPCR STR Setup System Getting Started Guide For more information Chapter 1 The HID EVOlution gPCR STR Setup System Table3 Required materials continued Material Sourcet Graduated 5 mL self standing transport tubes VWR 89005 596 master mix vials with conical bottom T4 Eo buffer 10 mM Tris HCl pH 8 0 and 0 1 User supplied or Teknova T0233 mM Na EDTA Degassed deionized water MLS MicroAmp optical 96 well reaction plate with Applied Biosystems PN 4306737 or N8010560 or without barcode RNase free microfuge tubes 1 5 mL certified Applied Biosystems PN AM12400 or equivalent DNase and RNase free Recommended sources Equivalent materials from other suppliers can be used after appropriate validation studies by the user laboratory Disposable tips that have not been certified by Tecan may not yield the same liquid handling performance For the MSDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all re
151. rst position in the extracted DNA sample plate or the first extracted DNA sample tube is always placed in well Al of the STR PCR reaction plate See Figure 6 below and Figure 7 on page 63 for STR PCR reaction plate layouts At the end of the STR PCR setup run the HID EVOlution system generates a report that lists the position of each DNA sample in the extracted DNA sample plate or tubes and in the STR PCR reaction plate After STR PCR amplification you transfer the PCR products from the STR PCR reaction plate to a CE plate for processing on the 3130 3130x Genetic Analyzer To make the plate to plate transfer easier the STR PCR reaction plate layout mirrors that of the CE plate layout For example the HID EVOlution qPCR STR Setup System leaves six empty wells designated as LDR in the STR PCR plate as placeholders for the allelic ladder sample replicates in the CE plate Figure 6 STR PCR plate layout for all AmpF STR kits except for the Yfiler kit PTC Amplification positive control control DNA NTC Amplification negative control TE buffer LDR empty wells indicating where allelic ladder sample replicates will be placed in the CE plate The positive and negative control volumes are 10 ul for 25 pL reactions and 20 pL for 50 pL reactions 62 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 7 Run Automated STR PCR Setup T For more information
152. s 3 Run CE CE and data analysis procedures are not included in this nad guide See the AmpF STR Kit User s Guides Perform Data Analysis 1 Prepare Sample Extract DNA from the forensic sample DNA extraction with the PrepFiler Forensic DNA Extraction Kits can be performed manually or automated on the HID EVOlution Extraction System Note Extraction procedures are not included in this guide For automated extraction procedures see the PrepFiler Automated Forensic DNA Extraction Kit Getting Started Guide HID EVOlution qPCR STR Setup System Getting Started Guide 89 Appendix C HID EVOlution gPCR STR Setup System Detailed Workflow Description 90 2 Perform quantitative PCR to quantify the total amount of amplifiable DNA a Prepare for automated qPCR reaction setup e e e Place extracted DNA samples in 96 well plates or 1 5 mL tubes Optional Create a qQPCR STR Sample file containing sample names and information for import to the HID EVOlution gPCR STR Setup System software Prepare Quantifiler kit reagents for use on the HID EVOlution qPCR STR Setup System Prepare the HID EVOlution qPCR STR Setup System b Run automated qPCR reaction setup on the HID EVOlution q PCR STR Setup System The HID EVOlution system e Optional Imports sample information from a qPCR STR Sample file Note You can prepare a qPCR STR Sample file before the run or if yo
153. s the 7500 SDS Real Time Software v 1 2 3 was used All runs were analyzed using the Manual analysis mode with the baseline set from 3 to 15 and the threshold set at 0 2 The R and slope values of the standard curve were calculated by the software and compared with previously observed ranges from the Quantifiler kit validations A slope value of 3 3 represents 100 efficiency of PCR A slope range of 3 0 to 3 6 is typically observed for the different AB TaqMan assays Average tolerances for acceptable efficiency are from 90 to 110 with a variation 2 After capillary electrophoresis samples were analyzed with the 3130x Genetic Analyzer using the Data Collection Software v 3 0 All samples were genotyped using the GeneMapper D Software v 3 2 using standard analysis methods see the GeneMapper ID Software v 3 1 User Guide PN 4338775 For statistical analysis box plot charts were created with the MiniTab Statistical Software version 15 which graphically summarized the distribution of the average replicate peak height for each sample concentration For other analysis Microsoft Excel was used to calculate averages and standard deviations and to produce bar charts HID EVOlution qPCR STR Setup System Getting Started Guide Appendix E Validation Experiments and Results Precision and reproducibility studies DNA standard dilution series preparation and reaction setup Precision and reproducibility studies DNA sta
154. s includes the required overfill volume Note Two replicates of the eight concentrations in the DNA standard dilution series are transferred to the qPCR reaction plate for a total of 16 DNA standard dilution series samples Plate prepared with BOTH Quantifiler Human and Quantifiler Y Human Male Quantification Kit You need two pre prepared DNA standard dilution series one for each kit For each of the two DNA standard dilution series you need 60 uL of each of the eight concentrations of diluted standard Note This includes the required overfill volume Note Two replicates of the eight concentrations in the DNA standard dilution series are transferred to the qPCR reaction plate for each of the two DNA standard dilution series for a total of 32 DNA standard dilution series samples If the pre prepared DNA standard dilution series does not meet the requirements then let the HID EVOlution system prepare the DNA standard dilution series for you using the standard and TE buffer volumes shown in step 2 on page 27 26 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 3 Prepare qPCR Reagents and Labware 3 Set up reagents 2 Ifthe HID EVOlution system is preparing the DNA standard dilution series use the table below to determine the required volume of undiluted Quantifiler Human DNA Standard and TE buffer Required Volumes for HID EVOlution system Prepared DNA Standard Dilution Series
155. sample shown as QTY in the 7500 Results file VOL Volume uL of extracted DNA sample added to a STR PCR reaction either 10 uL or 20 uL depending on the kit AMOUNT Target amount of DNA ng that you want in the STR PCR reaction AMOUNT is the Required Amount of DNA defined by the user in the Sample Normalization Adjustment window The usual range is 0 1 to 2 5 ng the default is 1 ng If CONC x VOL is Then AMOUNT No dilution is necessary if you select this sample for processing the HID EVOlution system transfers the maximum allowable volume of sample either 10 or 20 uL to the STR PCR plate AMOUNT The HID EVOlution system determines which dilution protocol to use to achieve an amount of DNA in the STR PCR reaction no more than 15 above or below the target amount that you specified See Dilution protocols with examples on page 98 2 Where necessary the system calculates the required dilution ratio DIL CONC x VOL AMOUNT where DIL is the Dilution Ratio for example a DIL of 1 548 represents a dilution ratio of 1 1 548 3 Based on the required dilution ratio the system selects one of the dilution protocols shown in Table 15 on page 100 for kits with a 10 uL addition or Table 16 on page 101 for kits with a 20 uL addition The maximum dilution in a one step dilution is 1 20 for kits with a 25 uL total reaction volume and 1 40 for kits with a 50 uL total reaction volu
156. stem Getting Started Guide Documentation This appendix covers E Related documentation 0 c eee eects 148 Applied Biosystems documentation 0 0 cece cece eee ees 148 Tecan documentation 2 0 0 cece ranee 149 B Obtaining information from the Help system 00 00 e eee 150 B Send us your comments 0 c cece teen eens 150 HID EVOlution gPCR STR Setup System Getting Started Guide 147 Documentation Related documentation Related documentation Applied Biosystems documentation 148 The following related documents are available for use with the system Part i gs Document numb Description HID EVOlution 4426903 Provides brief step by step procedures for qPCR STR Setup System setting up qPCR and STR PCR It is designed to Getting Started Guide help you quickly learn to use the HID EVOlution qPCR STR Setup System Quantifiler Kits 4344790 Includes information about manual quantitation Quantifiler Human DNA specific to your kit Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit User s Manual AmpF STR COfiler 4306116 Includes information about normalization and PCR Amplification Kit amplification specific to your kit User s Manual AmpF STR Identifiler 4323291 Includes information about normalization and PCR Amplification Kit amplification specific to your kit User s Manu
157. sults for each kit type as detailed in the Quantifiler Kits User s Manual Chapter 6 4 PN 4344790 Table 17 shows the observed slope and R ranges obtained from both the automated and the manual qPCR reaction setups A slope value of 3 3 represents 100 efficiency of PCR and a slope range of 3 0 to 3 6 is typically accepted to account for stochastic variation in samples The data indicate that the efficiency for Quantifiler Y Human Male ranged from 97 496 to 104 596 with a variance from the standard curve lt 1 6 The efficiency of the Quantifiler Human samples ranged from 92 6 to 112 6 with a variance from the standard curve of 1 3 Table 17 Slope and R values for Quantifiler Human and Y Human Male kits ND not determined HID EVOlution system prepared standards range observed for one 7500 instrument HID EVOlution system prepared standards range observed for three different 7500 Observed range published in the Quantifiler Kits User s Manual n 40 instruments n 75 Quantifiler Slope 3 052 to 3 370 3 052 to 3 512 2 9 to 3 3 Human kt R 0 991 to 0 999 0 987 to 0 999 9896 Quantifiler Y Slope 3 217 to 3 386 ND 3 0 to 3 6 Furman Male Mf R 0 984 to 0 991 ND 9896 HID EVOlution qPCR STR Setup System Getting Started Guide 107 E Appendix E Validation Experiments and Results Precision and reproducibility studies qPCR reaction setup
158. table and carriers to avoid cross contamination HID EVOlution qPCR STR Setup System Getting Started Guide Appendix F Automation Guidelines F STR PCR reaction setup automation guidelines STR PCR reaction setup automation guidelines The following are general guidelines for automating the dilutions for normalization mixing the STR PCR reaction mix for amplification and dispersing diluted samples and reaction mix into a reaction plate for the STR kits Before the Extract DNA then automated process 1 Quantify the concentration of each sample 2 Identify the target concentration and sample input volume for the STR kit to be used Target concentrations and input volumes STR kit Target mass ng a bia ac COfiler 0 5 to 1 25 20 50 Identifiler 0 5 to 1 25 10 25 MiniFiler 0 5 to 0 75 10 25 Profiler Plus 0 5 to 1 25 20 50 SEfiler Plus 0 5 to 0 75 10 25 SGM Plus 0 5 to 1 25 20 50 Yfiler 0 5 to 1 0 10 25 Prepare the After extracting DNA worktable 1 Place the DNA samples on the worktable 2 Place empty tube or vial for preparation of Master Mix on worktable Place two new 96 well plates on the worktable for use in dilutions 3 Place thawed STR PCR reagents on the worktable Centrifuge reagents before placing on worktable for maximum volume accessibility 4 Place T E buffer on the worktable 5 Place a new 96 well plate for the PCR reaction on the worktable
159. te For example if you are using the MiniFiler kit and your control DNA target concentration is 0 05 ng uL combine 30 uL of control DNA 0 1 ng uL with 30 uL of low T 9E buffer for a total volume of 60 uL Note The HID EVOlution qPCR STR Setup System does not dilute the control DNA instead the system transfers a set volume 10 or 20 uL depending on the kit of control DNA to the reaction plate AmpF STR Kit Recommended Target Concentration for Control DNA ng uL Minimum Required Volume of Diluted Control DNA uL Identifiler and Yfiler Kits 0 1 ng uL 60 MiniFiler and SEfiler Plus 0 05 ng uL 60 Kits Profiler Plus COfiler and 0 1 ng uL 70 SGM Plus Kits t This is the final recommended target concentration based on Applied Biosystems developmental validation studies and instrumentation The minimum required volume is the volume required by the experiment 10 uL or 20 uL plus a 50 uL dead volume HID EVOlution qPCR STR Setup System Getting Started Guide Set up reagents Chapter 6 Prepare STR PCR Reagents and Labware E b Place the required volume of diluted control DNA in an empty original control DNA tube with the narrow chamber and skirt or another tube with the identical shape IMPORTANT For proper liquid detection the diluted control must be placed into a tube with an identical shape to the original control DNA tube Using a 1 5 mL tube is not acc
160. te procedures for qPCR and STR PCR reaction setup E Onc time tasks chs gece nae EROS oka ge ae Red ase aos 10 Create detectors for the 7500 Real Time PCR System 04 10 Create an instrument protocol and results group for the CE instrument 11 Before each run Run maintenance scripts ees 12 Before each run Set up extracted DNA samples 2 0 0 0 000 e eae 13 Set up extracted DNA samples in a 96 well plate 0 0 0 0 ee eee 14 Set up extracted DNA samples in tubes 1 0 0 0 00 eee eee 15 Before each run Optional Set up sample information 17 About sample information 0 0 00 c ccc e 17 Options for entering sample information 000 2 eee eee eee 17 Create a qPCR STR Sample file n nonna tees 18 Guidelines Preventing contamination asese cece eee eee eee 20 For more information 0 ceser eaen ee he he nen nen 21 2 Chapter 2 Pre Run Procedures One time tasks One time tasks Complete the following tasks on each Real Time PCR and CE instrument before starting the automated procedures This will allow you to import the SDS Setup file generated by the HID EVOlution qPCR STR Setup System into the SDS software v1 2 3 e 7500 Results file s into the HID EVOlution gPCR STR Setup System software e CE Setup file generated by the HID EVOlution gPCR STR Setup System into the 3130 3130x instrument Data Collection Softwar
161. the SDS software Also shown as Qty in the EVOware software Sample Normalization window The volume of extracted DNA sample that is placed in the STR PCR reaction plate The volume 10 or 20 uL is pre defined in the EVOware software based on the AmpF STR kit that you use This volume is not user editable Guidelines for successful normalization 94 Provide an adequate volume of extracted DNA While the maximum sample volume required for any kit is 20 uL it is recommended that you check the samples for sufficient volume prior to dilution A 50 uL total volume is ideal for reliable liquid detection but you can use lower volumes IMPORTANT If there is insufficient sample for the required volume the entire volume will be aspirated and diluted according to the dilution protocol regardless of the deficient volume This deficiency could result in low or no profiles in the downstream analysis Setup the system When you load plasticware for an STR PCR setup run always load two pre dilution plates on the worktable to ensure there are adequate wells for dilution Select samples for processing During the STR PCR setup run review and adjust 1f necessary the information in the Sample Normalization Adjustment window see step 5 on page 59 When selecting samples for processing be aware of the limits to the HID EVOlution system sample normalization see page 95 HID EVOlution qPCR STR Setup System Gett
162. the Tecan HID EVOlution gPCR STR Setup System Application Manual Section 3 4 2 File Format for Plate Wells Tubes for details Manually enter sample information into the HID EVOlution software Run the appropriate EVOware software script then enter the information when you are prompted Use the Edit button in the Sample Information dialog box and manually add sample information See the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 Running a Script for details Note If you manually enter sample information for qPCR setup you will need to re enter the information for STR PCR setup To reduce setup time consider creating a qQPCR STR Sample file for use in qPCR and STR PCR setup as described below Note If you are using barcodes barcode scanning takes place before you import and or manually enter information You can manually enter sample information after barcode scanning and before or after importing a sample file Generally the information scanned imported entered last overwrites previous sample information Because initial software configuration can vary depending on customer requirements you may see different behavior with your system Open the qPCR STR Sample file template provided with the software CD a Select Start gt All Programs gt Accessories gt Notepad to open Microsoft Notepad IMPORTANT Use a text editor such as Microsoft Notepad to edit the sample input file D
163. tion For information on The standard curve refer to the ABI PRISM 7000 Sequence Detection System User Guide Quantifiler Kits User s Manual and the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute Quantification Getting Started Guide e Quantitation see the Applied Biosystems Quantifiler Kits User s Manual The 7500 Results file see the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide Setting up a plate document and plate document template see the Applied Biosystems Quantifiler Kits User s Manual HID EVOlution qPCR STR Setup System Getting Started Guide 43 H Chapter 5 Perform qPCR and Review Results For more information 44 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 6 Prepare STR PCR Reagents and Labware This chapter provides procedures to prepare reagents and labware for STR PCR The HID EVOlution x amplification reaction setup qPCR STR Setup a System W Review pre run checklist for STR PCR reaction setup 0 46 l EB Determine reagent volumes ssssslseeeee eene 47 About minimum required reagent volumes lsllsleslee less 47 Reagent volume tables 0 0 ec ent ete eee 47 Pre Run Procedures B Set Up reagents nis Cee o REOR CU HERR ERE XH OR Code HO 52 E Set up the labware on the worktable llls
164. tions during qPCR and STR PCR amplification may also be inconsistent because of the different control types being used This inconsistency is compounded by the sample dilution transfers that are required for normalization As a result tracking the location of the samples as they are transferred into the PCR reaction plates is imperative The HID EVOlution Software tracks sample names and positions during processing and generates a final report summarizing this information This precise information is required to properly process the samples Positional integrity of the samples was demonstrated by processing 80 previously typed human samples using the HID EVOlution system for quantification normalization and STR PCR set up For the quantification workflow the Quantifiler Human kit was used For STR analysis the Identifiler kit samples were in tubes and the SGM Plus kit samples were in a 96 well plate Both the sample tubes and the plate were labeled with barcodes For additional information about various plate layouts see About the qPCR reaction plate layout on page 36 for qPCR layouts and About the STR PCR plate layout on page 62 for STR PCR layouts Results The resulting STR profile for each sample was compared with the known profile A 100 concordance was observed The position of each profile was compared with its expected location in the qPCR and STR PCR plates and with the sample report generated by the software A
165. tration were prepared and placed in either a plate or in tubes The HID EVOlution system was then used to normalize the DNA concentration and prepare the STR PCR amplification reactions The experiment was repeated three times for extracted DNA in tubes and three additional times for extracted DNA in a 96 well plate Kit specific positive control and negative control reactions were prepared by the HID EVOlution system Peak height averages and standard deviations were calculated to evaluate the precision and reproducibility of the automated STR PCR setup Box and whisker plots were generated from the Identifiler data Figure 27 on page 115 and the SGM Plus data Figure 28 on page 116 using peak height averages from all kit specific loci amplified Representative STR profiles and peak height average data from the remaining validated kits are detailed in Other AmpF STR kits Supplemental precision and reproducibility studies on page 117 Figure 27 on page 115 is data from the Identifiler STR PCR setup The Identifiler kit assay setup is a 25 uL PCR reaction volume made up of 10 uL of normalized DNA and 15 uL of reaction mix The overall peak height average for all Identifiler PCR assay setup experiments regardless of the source vessel plate or tubes was 1239 RFU with a standard deviation of 509 not including the 0 025 ng samples For tubes only the peak height average was 1332 RFU 523 while for plates only the peak height aver
166. trifuge tubes QuantifilerY tubes esc 80 Quantifiler Human a 96 well plate QuantifilerHumanY plate esc AND Y Human Male Kits 2 total 1 5 mL microcentrifuge tubes QuantifilerHumanY tubes esc datog Two reactions are prepared for each of the 32 extracted DNA samples one reaction is prepared using the Quantifiler Human kit reagents and one reaction is prepared using the Quantifiler Y Human Male kit reagents 34 2 Follow the directions in the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 1 Running a Quantifiler Script IMPORTANT If you want the HID EVOlution qPCR STR Setup System to prepare the DNA standard dilution series make sure to select the Prepare Standards checkbox in the Reagent Information window of the script HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 4 Run Automated qPCR Setup 4 Perform post run tasks Perform post run tasks Take care of the qPCR reaction plate Clean up the instrument Remove the prepared qPCR reaction plate containing the qPCR reactions from the worktable Seal the qPCR reaction plate with MicroAmp Optical Adhesive Film PN 4311971 Place the qPCR reaction plate in a table top centrifuge with plate holders then centrifuge the plate at 3000 rpm for approximately 20 seconds to remove any air bubbles Remove the TE buffer trough and dispose of any remaining TE buffer
167. tup System Getting Started Guide 19 2 Chapter 2 Pre Run Procedures Guidelines Preventing contamination Guidelines Preventing contamination PCR assays require special laboratory practices to avoid false positive amplifications The high sensitivity of these assays may result in the amplification of a single DNA molecule To minimize false positives due to the presence of amplified material in your work area follow these recommended laboratory practices When possible maintain separate work areas dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products e Wear a clean lab coat not previously worn while handling amplified PCR products or during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect they are contaminated and before leaving the work area Use positive displacement pipettes with aerosol resistant pipette tips Never bring amplified PCR products into the PCR setup area Open and close all sample tubes and reaction plates carefully Try not to splash or spray PCR samples When pipetting from a kit component tube hold the cap of the tube in your gloved hand or be sure to set it down on a clean decontaminated surface Keep reactions and components sealed when possible Before opening sealed reagents or reaction tubes or plates centrifuge the tube or plate briefl
168. u used the HID EVOlution Extraction System for DNA extraction the HID EVOlution Extraction System generates a PCR STR Sample file csv output file that you can import for the qPCR or STR PCR reaction setup run See Options for entering sample information on page 17 Prepares the qPCR master mix Optional Prepares the DNA standard dilution series from the control DNA standard Transfers the master mix DNA standard dilution series and samples into a qPCR reaction plate and mixes the reaction components Generates a 7500 Setup file that can be imported into the 7500 Real Time PCR System SDS software v1 2 3 to set up the plate document for the qPCR run on the 7500 Real time System Note The HID EVOlution qPCR STR Setup System can prepare up to 80 qPCR reactions and 16 DNA standard dilution series reactions per run See Before each run Set up extracted DNA samples on page 13 c Run qPCR using the 7500 Real Time PCR System with SDS Software v1 2 3 e e Transfer the qPCR reaction plate to the 7500 instrument Import the 7500 Setup file to the SDS software Run qPCR Analyze and review the qPCR results HID EVOlution gPCR STR Setup System Getting Started Guide Appendix C HID EVOlution gPCR STR Setup System Detailed Workflow Description 3 Perform STR amplification to amplify specific STR loci in a single PCR amplification sample a Prepare for automated STR PCR amplificati
169. ual specific to your system e For details on the HID EVOlution qPCR STR Setup System see the Tecan HID EVOlution qPCR STR Setup System Application Manual Creating an analysis method instrument protocol and results group in the Data Collection software refer to the Applied Biosystems 3130 3130x Genetic Analyzers User Bulletin Using Data Collection Software v3 0 Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products PN 4363787 HID EVOlution qPCR STR Setup System Getting Started Guide 69 Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis For more information 70 HID EVOlution qPCR STR Setup System Getting Started Guide Appendix A Troubleshooting For troubleshooting problems with DNA yield see the Quantifiler Kits User s Manual and Table 12 below Setting up and running the automation instrument see the Tecan HID EVOlution qPCR STR Setup System Application Manual Table 12 Troubleshooting qPCR STR PCR results Observed problem Possible reason Suggested solution The DNA yield is low or The biological sample contains Confirm the correct reagent and instrument setup DNA is absent no or a low amount of DNA then re run the samples Reagents are missing or Confirm reagent and instrument performance improperly positioned on the Manually set up control DNA samples using the worktable same reagents and instrumentation to confirm ncorrect automated
170. uantifiler PCR Reaction mix 13 Empty 5 mL VWR tube for the master mix t The required number of tubes with the Quantifiler Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 pL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 pL plus a 50 uL dead volume If the volume of reagent in the first tube position 9 falls below 240 pL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid SHY UONBOYNUCN YNA eJeruueno suolyesnByuoy xooj g juebeey g xipueddy epinc peueis 6unjec uiejsAs ANIS H1S HOdb UONI AIH L8 Quantifiler Human and Y Human Male Kits with system prepared standards Quantifiler Human kit reagents Quantifiler Y Human Male kit reagents Figure 13 Quantifiler Human and Y Human Male kits with DNA standards prepared by the HID EVOlution system Legend 1 8 1 5 mL empty tubes for DNA standards 9 11 Up to three 1 5 mL tube s of Quantifiler Primer Mix Place the first tube in position 9 If you use more than one tube continue to position 10 then position 11 12 Quantifiler PCR Reaction mix 13 Empty 5 mL VWR tube for the master mix 14 Quantifiler Human DNA Stan
171. um number of extracted DNA samples that can be processed in a qPCR or STR PCR reaction setup run Quantifiler DNA Quantification Kit Number of extracted samples and controls Number of reactions in resulting qPCR reaction plate Other wells in resulting qPCR reaction plate Quantifiler Human Kit up to 80 up to 80 16 wells are used for the DNA standard dilution series 2 replicates of the 8 concentrations in the series Quantifiler Y Human Male Kit up to 80 up to 80 16 wells are used for the DNA standard dilution series 2 replicates of the 8 concentrations in the series Combined set up using both Quantifiler Human and Y Human Male Kits up to 32 up to 32 Quantifiler Human kit reactions up to 32 Quantifiler Y Human Male kit reactions 32 wells are used for the DNA standard dilution series 4 replicates of the 8 concentrations in the series Table 5 Maximum number of samples in a STR PCR reaction setup run AmpF STR PCR Amplification Kit Number of extracted samples and controls Number of reactions in resulting STR PCR reaction plate Other wells in resulting STR PCR reaction plate 1 well for amplification positive control 1 well for amplification negative control COfiler Identifiler up to 88 up to 88 MiniFiler Profiler Plus SEfiler Plus or SGM Plus Kits 6 empty wells Yfiler Kit up to 87 up to 87 i 2 w
172. ument protocol and results group for the CE instrument on page 11 1 On the HID EVOlution system navigate to the C HIDEVOlution_qPCRSTRfiles AB3130Input folder then locate the CE Setup file with the name that you noted at the end of the run STRplate_ lt rundate gt _ lt runtime gt txt or lt barcode gt _ lt rundate gt _ lt runtime gt txt 2 Copy and transfer the CE Setup file to a location where it can be accessed by the Data Collection Software computer 3 Inthe Data Collection Software a Select Plate Manager then click Import b Navigate to the CE Setup file that you copied from the HID EVOlution qPCR STR Setup System software to the CE instrument 3130 3130x Genetic Analyzer computer c Select the file click Open then click OK Prepare the CE Plate Refer to the applicable AmpFSTR Kit User Guide and the Applied Biosystems 3130 3130x Genetic Analyzers User Bulletin Using Data Collection Software v3 0 Protocols for Processing AmpF4STR PCR Amplification Kit PCR Products PN 4363787 68 HID EVOlution qPCR STR Setup System Getting Started Guide For more information Chapter 8 Perform STR PCR and Set Up Capillary Electrophoresis el For more information For information on Running STR PCR amplification refer to the appropriate 4mpF4STR Kit User S Manual Running CE see the Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide or the AmpF4STR Kit User s Man
173. ution gPCR STR Setup System Getting Started Guide Appendix G Safety e Chemical safety MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free MSDSS 24 hours a day To obtain MSDSs 1 Goto www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any ofthe following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer HID EVOlution qPCR STR Setup System Getting Started Guide 143 Appendix G Sa
174. ution Protocols Dilution protocols with examples Table 15 Dilution protocols for 10 pL transfer Columns 1 and 2 apply only for a DNA target input amount of 1 ng If your sample concentration is First Dilution Mixture D1 Second Dilution Mixture D2 Volume D1 between Then the or D2 dilution added to ratio is Volume of Volume from SIR FCR Min Max extracted TE added to D1 added to TE added to reaction ng pL ng pL DNA added D1 uL D2 uL D2 uL plate uL to D1 pL 0 000 0 115 1 1 Sample is transferred directly from the extracted DNA plate or 10 tube to the STR PCR plate 0 115 0 155 1 1 3 18 55 6 45 0 0 10 0 155 0 209 1 1 8 11 9 0 0 10 0 209 0 279 1 2 5 8 16 11 84 0 0 10 0 279 0 369 1 3 2 6 16 13 84 0 0 10 0 369 0 490 1 4 3 4 66 15 34 0 0 10 0 490 0 649 1 5 7 3 5 16 5 0 0 10 0 649 0 857 1 7 5 2 66 17 34 0 0 10 0 857 1 143 1 10 3 27 0 0 10 1 143 1 500 1 13 3 3 37 0 0 10 1 500 1 846 1 17 2 2 32 37 68 0 0 10 1 846 2 297 1 20 2 38 0 0 10 2 297 3 097 1 27 2 38 29 68 10 32 10 3 097 4 174 1 36 2 38 22 00 18 00 10 4 174 5 581 1 50 2 38 16 32 23 68 10 5 581 7 385 1 64 2 38 12 32 27 68 10 7 385 9 796 1 86 2 38 9 32 30 68 10 9 796 12 97 1 114 2 38 7 00 33 00 10 12 97 17 14 1 150 2 38 5 32 34 68 10 17 14 22 98 1 200 2 38 4 00 36 00 10 22 98 30 86 1 270 2 88 6 66 33 34 10 30 86 40 76 1 360 2 88 5 00 35 00 10 40 76 54 00 1 4
175. within these limitations The default limits in the HID EVOlution system software are set to the maximum and minimum concentrations of the standard curve for the Quantifiler kits 0 023 to 50 ng uL Samples with concentrations outside these limits will not be processed unless you manually edit the concentration range or select the individual sample s for processing Table 14 Concentrations within the HID EVOlution system dilution range 10 uL transfer into STR PCR 20 uL transfer into STR PCR Target plate plate Amount ng Min ng uL Max ng pL Min ng pL Max ng pL transfer transfer transfer transfer 0 1 0 01 40 0 005 40 0 125 0 013 50 0 063 50 0 5 0 05 200 0 025 200 1 0 0 1 400 0 05 400 1 5 0 15 600 0 15 600 2 0 0 2 800 0 2 800 2 5 0 25 1000 0 125 1000 5 0 0 5 2000 0 25 2000 10 0 1 0 4000 0 5 4000 HID EVOlution qPCR STR Setup System Getting Started Guide 95 Appendix D Dilution Protocols About DNA normalization on the HID EVOlution system About DNA normalization on the HID EVOlution system How the HID EVOlution system determines when to dilute an extracted DNA sample How the HID EVOlution system 96 dilutes a sample 1 For each extracted DNA sample the system uses the following values to determine if the sample needs to be diluted before it is pipetted to the STR PCR reaction plate CONC Concentration ng L of the extracted DNA
176. workstation for a given run 1 2 200 pL disposable pipette tips DiTis 3 6 50 uL DiTis 7 Trough for T 9E 4 buffer 8 Chilled qPCR Reagent Block 9 MicroAmp Optical 96 Well Reaction Plate qPCR reaction plate with 96 well metal plate adapter 10 MicroAmp Optical 96 Well Reaction Plate if extracted DNA samples are in a plate with 96 well metal plate adapter 11 Tube racks S1 through S5 for DNA sample tubes if extracted DNA samples are in tubes 32 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 4 Run Automated qPCR Setup This chapter provides procedures for performing a qPCR reaction setup run The HID EVOlution B Run a qPCR setup Sctiplics us a e CRUCE deeds ioe c Cer 34 qPCR STR Setup System About script files esd add eee Hew Re ed SUIS D PSP Yd RN 34 R n a req PUTEM 34 EB Perform post run tasks 0 cece eee teen enn 35 Prachi Procedures Take care of the qPCR reaction plate 0 cece eee nee 35 Clean up the instrument 0 0 teens 35 B About the qPCR reaction plate layout 0 20 eee 36 E For more information llle e 37 Prepare qPCR Reagents and Labware Chapter 4 Run Automated qPCR Setup Perform qPCR and Review Results Prepare STR PCR Reagents and Labware Run Automated STR PCR Setup Perform STR PCR and Set Up Capillary Electrophoresis HID EVOlution qPCR STR Setup S
177. y approximately two seconds in a microcentrifuge to collect any residual tube contents from the sides and cap e Clean lab benches and equipment periodically with freshly diluted 10 bleach solution Note For the Freedom EVO workstation follow with copious amounts of water 20 HID EVOlution qPCR STR Setup System Getting Started Guide Chapter 2 Pre Run Procedures 2 For more information For more information For information on Tecan s numbering system for plate wells and tube racks see the Tecan HID EVOlution gPCR STR Setup System Application Manual Section 3 4 5 Definition of Positions in Multi Well Racks and Plates Preparing a sample input file refer to the Tecan HID EVOlution gPCR STR Setup System Application Manual Section 3 4 2 File Format for Plate Wells Tubes and Section 3 4 5 Definition of Positions in Multi Well Racks and Plates Manually entering sample information refer to the Tecan HID EVOlution qPCR STR Setup System Application Manual Section 5 3 Running a Script Barcode specifications for use on the Freedom EVO instrument refer to the Tecan Freedom EVO Operating Manual Section 3 5 6 Positive Identification PosID and the Tecan HID EVOlution M qPCR STR Setup System Application Manual Section 4 6 Barcodes HID EVOlution qPCR STR Setup System Getting Started Guide 21 2 Chapter 2 Pre Run Procedures For more information 2
178. y studies eo ERE ER ARE EP ee S 114 Other AmpF STR kits Supplemental precision and reproducibility studies 117 Identifiler and SGM Plus STR PCR amplification reaction setup accuracy study 122 Complete system check and precision study 000 cece eee eee eee 126 Concordance and position ID confirmation study 0 00 e eee eee 131 Contamination study 2 ee eee gi weer Re ER ede ee Wee SE eee Reale 132 Conclusion ected Mot a hate ES Bae Rh eS Sas rh nad AUS IE hE IEE Bs 133 References cios 040028 be ae baie ae Gade oh ee Pani e S EC M 134 Automation Guidelines cc eee 135 qPCR reaction setup automation guidelines llli 135 STR PCR reaction setup automation guidelines llle 137 Contents Appendix G vi Safely ia at dieuie seated se o ree xem Es aeue adsis uc dt 141 Ghemical safety o tec i ead Aere were LT e be Pe De ED bet s 142 General chemical safety 00 00 cece nes 142 MSDSS xxu pude oy Seo nk a dtd aad t UN RR a ee Ge dS 143 Chemical waste safety eii reau nuaa a a e a a a eee eee 144 Biological hazard safety i un a a A EE EA N nh 145 Safety alerts o aE Leathe ied a a E e Bet Panes bad cia E xeu eS 146 Docurneritatlor ns ou acon d x ta a eaaeeeeoe dere algae es 147 Related documentation csere riei Iru a i a ET EEA eee eee 148 Obtaining information from the Help system 0 00 eee 150 Send us your comments cece ete eee 150 lr e
179. ystem Getting Started Guide 33 4 Chapter 4 Run Automated qPCR Setup Run a qPCR setup script Run a qPCR setup script About script files Run a script To begin automated qPCR or STR PCR reaction setup on the HID EVOlution qPCR STR Setup System you must run a script Scripts contain the workflow instructions defined for a specific instrument type the Freedom EVO 150 and 200 instrument worktable configuration and Freedom EVOware software version to automate a specific procedure For example there are six scripts available to set up qPCR reaction plates using the HID EVOlution system see table below and 14 scripts available to set up a STR PCR reaction plate see Run STR PCR setup on page 58 After performing all tasks including maintenance in the Review pre run checklist for qPCR reaction setup on page 24 use the following procedure to begin automated qPCR reaction setup 1 Select the appropriate EVOware software script for the Quantifiler kit s and plasticware that you are using Freedom EVOware software script selection for qPCR reaction setup If you are using the Maximum number of and the sample DNA is in use the script extracted DNA kit s samples per run Quantifiler Human Kit a 96 well plate QuantifilerHuman plate esc 80 1 5 mL microcentrifuge tubes QuantifilerHuman tubes esc 80 Coaster Y Human Male a 96 well plate QuantifilerY plate esc 80 i 1 5 mL microcen

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HID EVOlution™ - qPCR/STR Setup System Getting Started Guide