Elucigene® QST*R-PL (Pregnancy Loss) Assay Instructions for Use
Contents
1. Run AB3130A 2014 04 17 16 13 0057 Run_AB3130A_2014 04 17_16 13_0058 Run_AB3130A_2014 04 17_16 13_0059 Run_AB3130A_2014 04 17_16 13_0060 J Run_AB3130A_2014 04 23_10 46_0067 1 Run_AB3130A_2014 06 10_15 50_0100 _ Run_AB3130A_2014 06 13_15 16_0102 J Run_AB3130A_2014 06 16_12 46_0103 can d Run_AB3130A_2014 06 17_10 38_0105 Ly Run_AB3130A_2014 06 17_16 06_0106 1 Run_AB3130A_2014 06 18_09 12_0107 Run_AB3130A_2014 06 19_17 33_0110 J Run_AB3130A_2014 06 20_12 27_0111 J Run_AB3130A_2014 06 20_15 57_0112 Run_AB3130A_2014 06 20_15 57_0113 Run AB3130A 2014 06 20 15 57 0114 ull Run AB3130A 2014 06 23 12 09 0115 a ui Run AB3130A 2014 06 23 14 33 0116 Run AB3130A 2014 06 23 16 28 0117 amp uU Run AB3130A 2014 06 24 14 53 0118 J Run AB3130A 2014 06 24 14 53 0119 4s Run AB3130A 2014 06 25 13 54 0120 Run AB3130A 2014 06 25 13 54 0121 NEIN ER EE T7777 CTS AN6XYB1EN 001 Jan 2015 Page 13 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Importing QST R GeneMapper Analysis Settings in GeneMapper Manager It is necessary to import the QST R settings for GeneMapper This process is controlled through the GeneMapper Manager interface QST R GeneMapper settings are available from the Elucigene website www elucigene com product category rapid aneuploidy analysis p fo o gt Open GeneMapper Manager by clicking on the icon Select the2. 1 Combine 6 85yl of size standard with 250ul Hi Di Formamide and mix thoroughly sufficient mix for 16 wells Dispense 15ul of the mix into the required number of wells of a 96 well optical plate 2 Add 3ul of test sample PCR product to the size standard mix from step 1 already dispensed into the plate and mix using the pipette Seal the plate 3 Denature the PCR product dispensed into the optical plate on a thermal cycler using the following parameters 94 C for 3 minutes linked to 4 C for 30 seconds 4 Centrifuge the plate at 1 000g for 10 seconds to remove any bubbles in the wells and load onto the Genetic Analyzer Note t is essential that unused wells i e wells in which No DNA sample is loaded are still loaded with Hi Di Formamide to ensure that the capillaries do not dry out Post PCR Data Analysis ABI3130 GENETIC ANALYZER Create a sample sheet using the 3130 data collection software with the following settings e Sample Name this must be the same sample specific name or number Run owner select the default owner for lab e Run Protocol QSTR contains QST R 3130 run module see below Note It is necessary to create a run module detailing the instrument settings and subsequently assign this to a Run protocol in which Dye set G5 has been selected For more information on creating run modules please refer to your instrument user manual AN6XYB1EN 001 Jan 2015 Page 8 of 37 Elucigene amp QST R PL Pr
3. Figure 17 Run Wizard Template Selection Window Run Wizard Template Selection Set the template of the project f Select an existing template or create one A AFLP Template Name QSTR PL IM Panel gSTRPL gt y z il OSTA PL 44 Fragilex Ug 4 LOH Size Standard sso e m een Standard Color Dane i v 4 mPCR Analysis Type Fragment Animal v Next gt gt Cancel AN6XYB1EN 001 Jan 2015 To access the Run Wizard simply click on the Run Project icon in the main toolbar Select the Panel Size Standard Standard Colour and Analysis Type as shown in Figure 17 Page 26 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Run Wizard Data Process The Data Process window of Run Wizard allows the user to select the peak filtering parameters 1 Select the appropriate analysis settings in the Data Process window as shown in the figure below Figure 18 2 Click Next to continue Note The analysis range setting within the raw data analysis bow will vary depending on the Polymer used during data collection The operator should select a start data point value that includes the 75bp size standard peak Figure 18 Run Wizard Data Process Window F Run Wizard xs Data Process Fragment Animal Analysis Set data process options Raw Data Analysis Allele Call j Auto Range frame tal M Auto Range bps 0 Y
4. Analysis Methods tab and then click the import button Navigate to and import the required QST R Analysis Settings file s 8130 or 3500 Repeat the process selecting the appropriate tab and importing the corresponding file for e Table Settings e Plot Settings e Size Standards Note Cluster Plot Settings Matrices SNP Sets and Report Settings do not require file imports Importing QST R PL Panel settings in Panel Manager It is necessary to import the QST R PL panel and bin settings for GeneMapper This process is controlled through the Panel Manager interface QST R PL specific GeneMapper panel and bin settings files are available from the Elucigene website www elucigene com product category rapid aneuploidy analysis Open Panel Manager Program by clicking on the icon Click Panel Manager on the left navigation window Panel Manager will now appear highlighted in blue Select File Import Panels Navigate to and import the GeneMapper panel file QSTRPL Panel txt Figure 3 The panel file will now be displayed on the left navigation window Click on the panel file ensuring that it is highlighted blue Select File Import Bin Set Navigate to and import the GeneMapper bin file QSTRPL Bins txt Figure 4 Click Apply then click OK AN6XYB1EN 001 Jan 2015 Page 14 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Figure 3 Importing QST R PL Panel File Yr Edit Ana
5. ELUCIGENE and QST R are trademarks of Delta Diagnostics UK Ltd GENEMARKER is a trademark of Softgenetics Corporation GENEMAPPER GENESCAN NED VIC PET POP 7 LIZ and HI DI are trademarks of Life Technologies Corporation Note to purchaser Limited License This product is sold pursuant to an agreement with Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use only the purchased amount of the product and its components for human in vitro diagnostics solely for the indication described in the accompanying instructions for use For information on obtaining additional rights to use this product or its components please contact outlicensing lifetech com Copyright 2014 Delta Diagnostics UK Ltd AN6XYB1EN 001 Jan 2015 Page 37 of 37
6. SNP Sets Report Settings Projects Analysis Methods Table Settings Plot Settings Analysis Method Editor Microsatellite 2S pnt General Allele Peak Detector Peak Quality Quality Flags E Peak Detection Algorithm Advanced X Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Partial Range All Sizes w B 50 R 50 E Start Pt 2000 tart Size 0 zona e G 50 P 50 Stop Pt 18000 Stop Size 000 Y 50 O 20 Smoothing and Baselining Min Peak Half Width 2 pts Smoothing None Light Polynomial Degree Heavy Peak Window Size 15 pts Baseline Window 51 pts Peak Start 0 0 a m Peak End 0 0 2nd Order Least Squares 3rd Order Least Squares Size Standard Normalization Cubic Spline Interpolation a g Local Southern Method Global Southern Method Note For 35XX series te data collection normalization only Factory Defaults J cance AN6XYB1EN 001 Jan 2015 Page 16 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use To find the correct analysis range for your laboratory 1 6 From the main GeneMapper window double click on the imported Run Folder to view the list of fsa files it contains Select an fsa file Clicking the Raw data tab will display the electropherogram of the raw data Using the first peak of the size standard e g 75bp of GS500LIZ as a guide select on a data point approximately 100 data points larger Figure 6 Thi
7. 10000 j Start s g End 520 4 iw Smooth Enhanced Smooth Peak Detection Threshold Iw Peak Saturation Baseline Subtraction Min Intensity 50 1 Max Intensity E0000 3 5 Enhanced Baseline Subtracti Enhanced Baseline Subtraction Percentage Global Max iw Pul up Correction V Spike Removal Local Region gt 35 Local Max Size Call j Stutter Peak Filter 7z Iw Plus amp Filter Local Southern Cubic Spline Large Size Left 64 Right 32 Load Default Save Default I Note For 3500 data increase the Minimum Intensity to 150 lt lt Back Cancel AN6XYB1EN 001 Jan 2015 Page 27 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Run Wizard Additional Settings There are no additional settings required when performing QST R analysis Figure 19 1 Click OK to continue Figure 19 Run Wizard Additional Settings Run Wizard Additional Settings Fragment Animal Analysis Set additional options related to the different analysis type Allelic Ladder NONE Allele Evaluation Peak Score Reject lt 1 00 Check 7 00 Pass a 30 x Back i Cancel Data Processing Box After clicking OK in the Run Wizard Additional Settings box the Data Processing box appears Figure 20 The raw data is processed and sized then the filtering parameters and the selected QST R Panel are applied 1 Click OK in
8. Exit LA D135305 iz AMEL xg TAFIL zz SRY n 4 Ej Samples GH 12 Run AB3130 2014 B11_Run_AB31304_2014 J B12_Run_AB31304_2014 Cil Run AB3130 2014 C12 Run AB3130 2014 i 4 011_Run_AB31304_2014 D12 Run AB3130A 2014 Ell Run AB31304 2014 E12 Run AB3130A 2014 Fl Run AB3130A 2014 F12 Run AB3130A 2014 G11 Run AB31304 2014 H11 Run AB3130A 2014 Panel Name QSTR PL Ploidy 2 3 Navigate to and import the panel QSTR PL xml 4 Repeat the process as required for other relevant panel files AN6XYB1EN 001 Jan 2015 Page 25 of 37 Processing Data Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use After the raw data files have been uploaded to GeneMarker they are ready to be processed The processing step includes application of a sizing standard filtering of noisy peaks and comparison to a known allelic panel if desired GeneMarker combines all these steps in one simple tool called the Run Wizard Figure 17 To access the Run Wizard simply click the Run Project icon in the main toolbar Run Wizard creating a Run Template It is necessary to create a run template the first time this software is used to analyse QST R PL data This is done through the Run Wizard 1 2 Assign a Template Name e g QSTR PL Click Save to store the template for future analyses Click Next to continue
9. PL Pregnancy Loss Assay Instructions for Use Capillary Electrophoresis Capillary Electrophoresis GeneScan 500 LIZ size standard ABI Cat No 4322682 POP 7 Polymer ABI Cat No 4352759 DS 33 dye set G5 matrix standard ABI Cat No 4345833 10x Genetic Analyzer Buffer ABI Cat No 402824 and Hi Di Formamide ABI Cat No 4311320 Applied Biosystems ABI 3130 and 3500 Genetic Analyzers with current Data Collection software 36cm capillary array 50cm capillary array for 3500 Genetic Analyzer 96 well optical plates 96 well septa 96 well cassettes Data Analysis One of the following data analysis software packages is required GeneMapper 4 1 Applied Biosystems Inc or above or GeneMarker 1 65 SoftGenetics LLC or above Additional Elucigene QST R Documentation These Instructions for Use include a basic section on interpretation of the results obtained in addition to a guide to software analysis with both the GeneMapper and GeneMarker packages A supplemental Guide to Interpretation with examples and glossary are available from the Elucigene website www elucigene com DNA Extraction A DNA Extraction method to yield PCR amplifiable quality DNA at a concentration of 0 5ng ul to 4ng ng ul DNA Concentration Using the recommended PCR conditions and sample injection settings stated in the capillary column run module page 9 optimal results are obtained with an inout DNA amount of 2 5ng However interpretable results are ob
10. Page 23 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Figure 14 Raw Data Analysis Window Sample File Tree Report Table Synthetic Gel Image Electropherogram GeneMarker Untitled liz File View Proj ct Applications Tools Help a gt HEH S BH 9 9 P E E B IA Maker None Ty E Tee AAJ dus Alle Cal pes 03 17 QSTR QC 22102009 1 aos 17 osre oc 4 NUT B 803 18 QSTR QC 22102009 z pos 1 osTR oc 4g BB C03 19 QSTR QC 22102009 s cos 19 osrR oc 48 B D03 20 QSTR QC 22102009 a pos zo asa ac 3 s jns 21 osTn oc 4g e ros_z2_osta_oc 4B E03 21 QSTR QC 22102009 F03 22 QSTR QC 22102009 G03 23 QSTR QC 22102009 H03 24 QSTR QC 22102009 on Onset WN 02 14 QSTR QC 22102009 mo ur G02 15 QSTR QC 22102009 L13 QSTR QC H02 16 QSTR QC 22102009 ao eRe 150 200 250 900 350 400 450 soo 22__OZ_26_0stm_oc_ 4i A03 17 QSTR QC 22102009 A fsa x jm m3s1187 1302151409884 M1 3S259C O21 1 44299 858153562065534 200 300 400 500 B B B B BB E02 13 QSTR QC 22102009 e Hos 24 asTR oc 44g BF B B Page Up Page Down AN6XYB1EN 001 Jan 2015 Page 24 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Importing QST R PL GeneMarker Panel Settings It is necessary to import the QST R panel settings for GeneMarker This process is controlled through the Panel Editor interface QST R PL GeneMarker
11. Set Run Name m Add Samples to Project p Edit View Files GM Database Samples To Add MBRun_AB3130A_2014 03 19_15 10_0020 Gj Run AB3130A 2014 03 19 15 10 0020 3j Run AB3130A 2014 04 10 09 20 0032 A01 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa 5 Jj Run AB3130A 2014 04 14 14 24 0033 A02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa Run AB3130A 2014 04 14 14 24 0034 L B01 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa a J Run AB3130A 2014 04 14 14 24 0035 L 4 amp B02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa L Run_AB3130A 2014 04 14 14 24 0036 4 CO1 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa a Run AB3130A 2014 04 14 14 24 0037 L4 CO2 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa a i Run AB3130A 2014 04 14 14 24 0038 L4 D01 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa Run AB3130A 2014 04 15 09 00 0039 L4 D02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa l Run AB3130A 2014 04 15 15 50 0043 4 E02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa 7 Run_AB3130A_2014 04 17_08 58_0048 H F02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa Li Run AB3130A 2014 04 17 08 58 0049 G02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa Run AB3130A 2014 04 17 08 58 0050 i H02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa J Run AB3130A 2014 04 17 08 58 0051 J Run AB3130A 2014 04 17 08 58 0052
12. T13 XY 2 were T13 XX 7 were Monosomy X 8 were triploid for all chromosomes tested One sample gave an uninformative result due to homozygosity within the marker set This sample was subsequently analysed using QST R 18 and was shown to be T18 XX All analysable results showed 100 concordance with results previously obtained by alternative established methods AN6XYB1EN 001 Jan 2015 Page 12 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use GeneMapper Analysis Guide Note The following GeneMapper screen shots are taken from GeneMapper v5 0 Importing and Analysing QST R Files 1 Open the GeneMapper Program file 2 Click in order to add data files to a new project Navigate to where the raw fsa data files are stored highlight the appropriate data files and click the Add to List gt gt button 3 Therun folder will now appear in the Samples to Add window Double clicking on the run folder icon in this window will show each fsa file to be imported Samples are then added by clicking at the bottom of the screen The data files now appear within the GeneMapper main screen figure 2 Figure 2 Samples ready to add to project File Edit Analysis View Tools Help oo E ae m B A gt Table Setting QSTR Table Settings v02 Le De I1 Samples Status Sample File Sample Mame Sample ID Comments Sample Type SFN Analysis Method Panel Size Standard Matrix SNP
13. al bul ulis dis sull l Figure 22 Trisomy Analysis Settings Box x Trisomy Analysis Settings Analysis by C Classic BPG C Aneuploidy Peak Height 50 Height Ratio gt 30 00 Max Quantification by Peak Height Peak Area Trisomy Ratio j Shorter Length Longer Length Apply Linear Correction Trisomy lt 10 80 or 1 40 Iw Inconclusive Range 10 65 to lt 0 80 or gt 1 40 to lt 1 80 Save Parameters when Save Report AN6XYB1EN 001 Jan 2015 Page 29 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Trisomy Analysis Settings Within the Trisomy Analysis settings box two tabs are available 1 Analysis tab 2 Statistics Plot tab Analysis Tab The Analysis tab provides threshold setting options for Trisomy analysis Ensure that BPG is selected in the analysis box and the following settings displayed Peak Height 50 Minimum height of 50 for peaks to be called 150 if using 3500 data Height Ratio 3096 Maximum percentage of the main peak the second peak must reach in order for two alleles to be identified Quantification by Peak Area Shorter Length Longer Length selected Trisomy Ratio thresholds are 0 80 1 40 Apply Linear Correction de selected Click OK Trisomy Analysis Window The Trisomy Analysis window Figure 23 allows the operator to review QST R sample data and display the ratio of peaks for each marker
14. from Template in the Dashboard ensure the correct Instrument Protocol for QST R has been assigned see above AN6XYB1EN 001 Jan 2015 Page 9 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Analysis and Interpretation of Results It is recommended that each laboratory develops its own interpretation and reporting procedures and criteria Best practice guidelines for QF PCR have been documented by the UK s Association for Clinical Genetic Science and are available for reference at www acgs uk com PCR products are observed as a 5 dye labelled system using filter set G5 Filter set G5 detects the 6 FAM blue VIC green NED yellow and PET red labelled fragments plus the Size Standard marker labelled with LIZ orange on an electrophoretogram and in the GeneMapper or GeneMarker program A QST R Guide to Interpretation is available from the Elucigene website www elucigene com Important Note different combinations of instrument polymer and size standard may cause the size calling to vary slightly During validation of the kit users should check that the default bin settings result in accurate peak labelling and adjust if necessary In case of any difficulty please contact Technical Support techsupport elucigene com for advice General analysis guidelines for QST R PL 1 The negative control should show no sharp peaks within the read range of 100 to 510bp 2 The positive control must show th
15. panel settings are available from the Elucigene Website www elucigene com product category rapid aneuploidy analysis 1 Open Panel Editor from the Tools drop down menu Figure 15 Figure 15 Selecting Panel Editor File View Project Applications Tools Help oe xmre UZC OBB De Make Plows co UL Size Template Editor Alele C Macromolecules Gel image Output Trac Export Electropherogram EL 0141 ll Magic Wizard 4111 Run 831304 2014 1C Show Last Event EBIBIJIBIBISIBIDIDIBIBIDID E rommmmoononoovoo rr5 E 100 110 120 130 40 150 60 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 SOD 510 ATI Fun AB31304 2014 10 24 15 12 0293 2014 10 24 tes AMEL ars sav 0225886 K FES FPS I 0155322 01351 02151442 LI 0185819 0165753 ps LC a 90 100 110 12 t 140 150 69 170 180 1900 200 2 220 230 240 6 Q t TA ot we IT X M 40 5 36 370 5 100 400 4130 420 430 44 450 46 470 489 490 500 5 0 52 Page Up Page Dow 2 Select Import Panels from the File drop down menu figure 16 Figure 16 Importing Panels Panel Edito Tools Help 3 Create New Panel Ira 7 Help X Delete Current Panel Marker lg Save Changes Ctrl S Save Changes with Signal Info Save As New Panel Gar Import Panels Import Pre defined Panels Import ABI Panels Import MRC Holland Panels Export Panel
16. the unlabelled peak You will get the option to add allele comment Click OK The peak will now be labelled with its size in base pairs and its peak area The table will automatically incorporate the newly labelled peak To remove a peak label left click on the peak label You will get the option to delete allele comment Click OK The peak label will now be removed The table will automatically remove the deleted peak data Copying Table Data 1 Highlight all rows with the table at the bottom of the plot window 2 Copy the selected rows by typing Ctrl C keys AN6XYB1EN 001 Jan 2015 Page 19 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use QST R PL Report Template The associated QST R PL report template can be used to determine the peak ratios for a marker and assist in analysis of data The QST R PL specific Report Template is available from the website www elucigene com product category rapid aneuploidy analysis 1 Open the QST R PL Report Template file QSTR PL Report Template xlsm 2 Ifthe Report Template displays a warning indicating that macros have been disabled click the Enable Content button to enable macros Figure 10 Figure 10 Enabling macro function in QST R PL Report Template HOME INSERT PAGE LAYOUT FORMULAS DATA REVIEW VIEW 10 Clipboard Fu Alignment 0 v SECURITY WARNING Macros have been disabled Enable Content 3 Ifa security warning is
17. to the left The data table can be printed or saved as a new file for each specific sample 6 In order to process subsequent samples it is important that all data is fully cleared from the report template In order to do this click the CLEAR DATA button located underneath the raw data table within the QST R PL Report Template New sample data can now be imported following the procedure outlined above AN6XYB1EN 001 Jan 2015 Page 21 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Scoring the Report 1 Trisomy is determined by either a wo peaks of uneven height due to one of the peaks representing two alleles which are common to both parents In this case the ratio between the two peaks will be classed as 2 1 or 1 2 Where A1 A2 will give a result in the region of 1 8 to 2 4 when the peak representing the shorter length allele is greater in area than the peak representing the longer length allele or where A1 A2 will give a result in the region of 0 45 to 0 65 when the peak representing the shorter length allele is smaller in area than the peak representing the longer length allele In both cases Ratio will appear in the Warning column b Three peaks of comparable height present The ratio of the peaks will be classed as 1 1 1 and their values fall within the normal range of 0 8 1 4 although for alleles separated by more than 24bp an allele ratio of up to 1 5 is acceptable In this case 3 Al
18. 10bp chromosomes and can be used to determine the presence or absence of a Y chromosome and represents the relative amount of X to Y sequence Please note that on rare occasions amplification failure due to mutation of the AMEL Y sequence has been reported TAF9L is an invariant paralogous marker with sequences on chromosomes 3 and X The chromosome 3 specific peak 116bp representing 2 copies of chromosome 3 can therefore be used as a reference peak to assist in the determination of the number of X chromosomes present 121bp peak Analysed in combination with Amelogenin and the other sex chromosomes markers it is particularly useful in the detection of sex chromosome aneuploidy In a normal female the markers should fall within a ratio window of 0 8 to 1 4 In a normal male the markers will give a ratio 21 8 Further details on the interpretation of the TAF9L marker can be found in the Guide to Interpretation The Y specific marker SRY will give a single peak in normal males and will not amplify in normal females AN6XYB1EN 001 Jan 2015 Page 11 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Performance Characteristics INTERNAL VALIDATION 125 fetally derived tissue samples were tested using Elucigene QST R PL Of these 34 were normal XY 40 were normal XX 5 were T22 XY 1 was T22 XX 3 were T21 XY 4 were T21 XX 3 were T18 XY 5 were T18 XX 6 were T16 XY 2 were T16 XX 1 was T15 XY 1 was T15 XX 2 were
19. Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Elucigene 9 Diagnostics Elucigene QST R PL Pregnancy Loss Assay Instructions for Use Cat Code AN6XYB1 25 tests For In Vitro Diagnostic Use ud by Elucigene Diagnostics Citylabs Nelson Street Manchester M13 9NQ For Sales Customer Service and Technical Support T 44 0 161 669 8122 F 44 0 161 669 8129 E enquiries elucigene com E techsupport elucigene com Elucigene Diagnostics is the trading name of Delta Diagnostics UK Limited a company registered in England and Wales registration number 8696299 AN6XYB1EN 001 Jan 2015 Page 1 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Elucigene QST R PL Intended Use For the routine in vitro diagnosis of the six most common autosomal trisomies associated with pregnancy loss trisomy 13 Patau syndrome trisomy 15 trisomy 16 trisomy 18 Edwards syndrome trisomy 21 Down syndrome and trisomy 22 The kit also includes X and Y chromosome markers and the TAF9L marker for the determination of sex status The method employed by the Elucigene QST R PL kit is the QF PCR Quantitative Fluorescence Polymerase Chain Reaction technique The device is intended to be used on DNA extracted from either fetal material obtained post miscarriage or whole blood of fetal origin The intended target population is individuals who have experienced a spontaneous miscar
20. Instructions for Use APPENDIX 1 Dye Labels The markers are labeled as follows NED SRY D22S686 D22S689 D16S2621 D155659 D21S1411 D13S628 See Appendix 2 for further details of the STR markers including size ranges AN6XYB1EN 001 Jan 2015 Page 33 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use APPENDIX 2 Table of Maker location Observed Heterozygosity allele size range Note the NED dye used in the kits is identified spectrally as a yellow dye It is conventionally displayed in black type for clarity D15S659 15q21 1 0 86 395 441 yellow D18S535 18q12 3 462 503 blue D18S386 18q22 1 353 440 green 291 332 blue D22S686 22q11 2 328 394 red 319 389 yellow 146 199 yellow 222 264 green D22S683 22912 3 152 223 blue AMEL Xp22 22 Yp11 2 TAF9 3p24 2 Xq 1 1 104 110 yellow 116 121 yellow Yp11 31 124 145 yellow Observed heterozygosities are based on number of alleles observed with Elucigene Diagnostics testing panel These figures may therefore differ from published data and may also vary according to the population being tested AN6XYB1EN 001 Jan 2015 Page 34 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use APPENDIX 3 GeneMapper Profile GeneMapper normal male profile showing relative positions of the markers detected by QST R PL A AN6XYB1EN 001 Jan 2015 Page 35 of 37 Elucigene amp QST R PL Pregnancy Lo
21. RE iu plis ig t sui 4 The plot window will display sample profile with the tabulated data Figure 9 A maximum of two peaks for each marker will be labelled automatically by GeneMapper If three alleles are present for a marker the third unlabelled peak should be manually labelled see Manual Editing of Profiles below Note Allele size ranges for each marker are based on previously observed data Rare alleles may fall outside the given marker size range and it may be necessary to adjust the bin set accordingly 5 It is recommended that the plot window has Single click editing activated To do this select Alleles set click editing and ensure that this option is checked AN6XYB1EN 001 Jan 2015 Page 18 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Figure 9 Sample Plot window displaying labelled trace data and correlating genotype table Samples Plot File Ed amp View Tools Alleles Plot Setting QSTR Plot Settings gt BD Fees 3 GN ER Boe wee ii i i db d I amp Sa e File B 8 8 E B B B 8 8 8 S ss Z ss ss Manual Editing of Profiles WARNING GeneMapper will only assign labels for up to 2 peaks per marker Manual editing of profiles may therefore be required i e when assigning labels to 3rd peaks if present or removing labels from stutter peaks To add a peak label left click on
22. aeu fosses fo p meme wsoss for 4 z arm EE 2 i 2 1 1 0 i on a O Oo oo Nm wo 50 200 300 350 400 450 500 1 250 cn 340 376 7331 6148 09 19 06 318 326 4787 4521 i ii 6775 7403 e elc NN 2 asz reseno T o fa 3 2 1 118 122 13145 6766 26 406 426 4g 200 250 350 400 450 500 ad Oj Cn j The GeneMarker report includes the following features e Report Header Contains information about the analysis project sample and parameters e Signature Box Date and initial space for report reviewers e Electropherogram Similar to the Trisomy analysis window displays all dye colours of the sample trace e Report Table Displays selected peak and marker values for the current sample Trisomy calls are highlighted grey An additional check column is provided for reviewer initials e Corrected Ratio Plot Contains the entire dataset s plot points for all markers in the dye colour Symbol shapes represent different markers and can be deciphered from the Symbol row in the Report Table Yellow filled symbols represent the current sample s data points Red outlined symbols represent trisomy calls Note The Corrected Ratio Plot appears on a second page for each sample only when Ratio Plot is selected in the Trisomy Print Report Settings box AN6XYB1EN 001 Jan 2015 Page 32 of 37 Elucigene amp QST R PL Pregnancy Loss Assay
23. and access the GeneMarker report There are a number of displays which assist the operator in data analysis They are Sample List Electropherogram Ratio Plot Report Table Figure 23 Trisomy Analysis Window Sample List Electropherogram Report Table VL Trisomy Analysis Raw fiato E amp ZA B QAQG6GEl _ r Ratio Plot For more detailed information regarding Trisomy analysis features and their use please refer to the GeneMarker Manual AN6XYB1EN 001 Jan 2015 Page 30 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use GeneMarker Report GeneMarker includes a report template that is compatible with Elucigene QST H kits To access the report click the Print icon located on the toolbar at the top left of the Trisomy Analysis Window im Sa This will open the Trisomy Print Settings window Figure 24 Trisomy Print Settings Window The Trisomy Print Settings detail the options for including and visualising sample data in the GeneMarker report Select the options as shown in Figure 24 Ensure that Custom Size Range is set to 98bp Start and 510bp End Figure 24 Trisomy Print Settings Window Trisomy Print Settings Samples Selected Samples Markers Ratio Plot v All Markers fe Show Population Selected Mark
24. dual samples 2 Click Open button in the Open dialog The files selected will appear in the Data File List field Figure 13 Figure 13 Samples added to the Data File list Open Data Files Data File List C SoftGenetics Gene Marker Datasets 4FLP frag_001_H01 fsa C SoftGenetics Gene MarkersD atasetsy FLP frag 002 _G01 fsa C SoftGenetics Gene MarkersDatasetsy amp FLP frag 3 F T fsa C SoftGenetics Gene Marker Datasets 4FLP Mrag_004_E01 fsa C SoftGenetics Gene MarkersD atasetsy FLP frag 05 DOT fsa C SoftGenetics Gene MarkersDatasetss amp FLP frag 05 COT fsa C SoftGenetics Gene MarkersDatasetssAFLP frag O07 BU T fsa R All C SoftGenetics Gene Marker Datasets AFLP frag_0O08_A01 fsa ve C SoftGenetics Gene MarkersDatasetsv amp FLP frag O03 HO3 fsa C SoftGeneticssGene MarkersDatasetss amp FLP frag 10 G 3 fsa C SoftGenetics Gene Marker Datasets 4FLP rag_011_FO3 fsa C SoftGenetics Gene Marker Datasets 4FLP rag_012_E03 fsa C SoftGeneticssGene MarkersDatasetsy FLP frag 013 DU3 fsa Add Folder C SoftGenetics Gene MarkersDatasetss FLP frag 14 CO3 fsa C SoftGenetics Gene MarkersDatasetss amp FLP frag 015 BOS fsa A Softienetics Gene Marker Datasets4Fl PMfran MA ANA fsa T Channels OK Cancel Remove Click OK button in the Open Data Files box and the samples will be uploaded to GeneMarker The software will then automatically open the Raw Data Analysis window Figure 14 AN6XYB1EN 001 Jan 2015
25. e expected results and all peaks must meet the criteria below 3 For analysis of DNA samples at least 1 peak should be observed for each marker tested The acceptable range for marker peaks analysed on the 3130 Genetic Analyzer is between 50 and 6000 relative fluorescent units rfus and for the 3500 Genetic Analyzers is between 175 and 32000 rfus Peak heights falling outside this range must not be analysed 4 Electrophoretograms of poor quality due to excessive bleed through between dye colours also known as pull up or electrophoretic spikes sharp peaks present in more than one dye should not be interpreted The PCR products should be re injected and re analysed 5 Analysis is performed by assessment of peak ratios A1 A2 where A1 is the peak area of the shorter length fragment and A2 is the peak area of the longer length fragment The resulting ratio is indicative of locus copy number For disomic chromosomes heterozygous markers should show two peaks with similar heights A complete analysis of chromosome copy number status is performed by comparison of peak area ratios 6 Heterozygous di allelic i e two alleles markers should fall within a ratio window of 0 8 to 1 4 However for two alleles separated by more than 24bp in size a ratio of up to 1 5 is acceptable Any values falling within this region are referred to as having a ratio of 1 1 If the ratio balance falls out of this window then it may be due to a number of fac
26. egnancy Loss Assay Instructions for Use 3130 RUN MODULE FOR POP7 POLYMER 36cm Capillary Module QSTR Parameter Name Oven Temperature Poly fill Vol 6500 38000 steps Current Stability int 0 2000 uAmps PreRun Voltage 15 kvolts Pre Run Time 1000 sec Injection Voltage 15 kvolts Injection Time 600 sec Voltage Number of Steps 100 nk Voltage Step Interval 60 sec Data Delay Time 3600 sec Run Voltage 15 kvolts Hun Time ABI3500 GENETIC ANALYZER 14000 sec A QST R Instrument Protocol needs to be created which can then be used for each QST R run Create the QST R Instrument Protocol through the 3500 Instrument Protocols library Ensure the following are selected e Run Module FragmentAnalysisbO0 POP7 Enter the settings detailed in the image below Edit Instrument Protocol QSTR Setup an Instrument Protocol Application Type Fragment v Dye Set 65 v Instrument Protocol Properties Capillary Length 50 v Run Module FragmentAnalysis50_POP Protocol Name OSTR Description Oven Temperature C 60 Run Voltage kVolts 19 5 Run Time sec 1300 PreRun Time sec 180 gt Advanced Options Close Polymer POP 15 Injection Voltage kVolts 3 0 15 Data Delay sec 1 Torun the samples create a sample plate by clicking on Create Plate
27. ers Show Sample Only Report T able W Show Allele Mame Iw Show Peak Height Area lw Show Peak Ratio J Show Trisomy Scores W Show Allele Iv Show Observation Electrapheragram Size Range Show Curent Size Range f Show All Markers Size Range f Show Custom Size Range start bp 88 End bn 51 0 Scale data to highest peaks within size range eb Ok x Cancel AN6XYB1EN 001 Jan 2015 Page 31 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Previewing the GeneMarker Report Click Preview to view the GeneMarker report Figure 25 From this window the operator can review and print each sample s peak data from across all markers providing a simple one or two page sample report Figure 25 GeneMarker Report Trisomy Analysis Report SoftGenetics Sample A11 Run AB3130A 2014 10 24 15 12 0299 2014 10 24 fsa Panek OSTRPL Classification Trisomy lt 0 80 or Trisomy gt 1 40 inconclusive gt 0 65 to 0 80 or 1 40to 1 80 maner Atee Atte Name Peak Area Peak Ratcneck observation ofs ozs UziSi fame 2 xv eese 2 150 200 250 300 350 400 450 50 D135305 3 1 1 1 464 480 484 5590768825910 D135325 3 1 1 1 316 320 324 7608 6844 6046 praszs 30 1 a82486490 2729940895 J OOOO Disex saamassa serrssszsoz0 O O pissists p teeter sezzees os pisses f2
28. leles will appear in the Warning column 2 To interpret a result as abnormal i e trisomy present at least two informative markers consistent with a tri allelic genotype are required with all other markers being uninformative It is not recommended to interpret a result as abnormal based on information from only one marker 3 Tointerpret a result as normal at least two informative markers consistent with a diallelic genotype are required with all other markers being uninformative A normal result indicates the normal complement of two for the chromosomes tested 4 Peak area ratios that fall between the normal and abnormal ranges are classed as inconclusive Inconclusive results may be resolved by using the single chromosome kits In the absence of any peak data for a marker Absent will be displayed in the warning column This warning will be routinely observed in the absence of Y chromosome markers AN6XYB1EN 001 Jan 2015 Page 22 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use GeneMarker Analysis Guide Note The following screen shots have been taken from GeneMarker v2 6 3 Adding Sample Files to GeneMarker Open the GeneMarker program file and when prompted select Open Data The Open Data Files box will appear Click the Add button The Open dialog will appear Navigate to directory containing raw data files 1 Select all files by CTRL A or use CTRL and or SHIFT keys to select indivi
29. ll samples should be considered potentially infectious No test method can offer complete assurance that HBV HCV HIV or other infectious agents are absent 3 Handling of samples and test components their use storage and disposal should be in accordance with the procedures defined by the appropriate national biohazard safety guideline or regulation 4 Inline with current good laboratory practice laboratories should process their own internal QC samples of known type in each assay so that the validity of the procedure can be assessed 5 f kit box is damaged there may be damage to the contents do not use the kit contact Customer Service AN6XYB1EN 001 Jan 2015 Page 3 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Symbols used on labels The symbols used on all labels and packaging conform to the harmonised standard ISO 15223 Manufacturer Number of tests See Instructions for Use Store below temperature shown Use before date shown Catalogue code Lot or batch number In Vitro Diagnostic Medical Device AN6XYB1EN 001 Jan 2015 Page 4 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Materials Provided Store all components below 20 C The Elucigene QST R PL IVD kit contains 450089 1 x 250ul Reaction Mix TA 404485 1 x 50ul Control DNA DC Sufficient for 25 tests Kit Preparation and Storage Upon opening the kit it is recommended tha
30. lysis View Tools Help SOR he BM BW BEB gt e wc QTR Tabie setings voz 8 biceo ef Project Figure 4 Importing QST R PL Bin File sey Project Samples co Bar Be GD n QTR Taie setna v02 ja 8 20 Sample Name Sample ID Comments Sample Type SFN a Fite Edit Bins View 4x Bi onset qstRewva 0 v E3esme a Panel Name 1 AN6XYB1EN 001 Jan 2015 Page 15 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Modifying the Analysis Parameter file It may be necessary to modify the default Analysis Ranges in the QST R analysis settings to account for local variance in run conditions The minimum analysis range will depend on the capillary and polymer being used during data collection To view current analysis settings 1 Open GeneMapper Manager by clicking on the icon 2 Select the Analysis Methods tab The Imported QST R file will be listed as QSTR Analysis v02 3 Click on QSTR Analysis v02 The row will now appear highlighted 4 Click the Open button and select the Peak Detector tab Figure 5 By default the analysis ranges are set to start at 2 000 and finish at 18 000 Figure 5 Analysis Ranges E GeneMapper Manager Cluster Plot Settings Matrices Size Standards
31. riage The results obtained from QST R PL kit can help determine the aneuploidy status of the fetus and is intended to be used in conjunction with other diagnostic procedures to support or discount the proposed clinical diagnosis The device is for Professional Use only within a molecular or cytogenetics laboratory environment Summary and Explanation Statistically 10 20 of all pregnancies end in spontaneous abortion miscarriage the majority of which occur towards the end of the first trimester Of these over 5096 of cases have been shown to be caused by a chromosome abnormality 1 primarily aneuploidy the most commonly noted are trisomies which account for 6096 of all chromosome abnormalities in miscarriage The most frequent trisomy found in products of conception POC is trisomy for chromosome 16 however trisomies for chromosomes 13 15 18 21 and 22 are also amongst the most common Other aneuploidies commonly seen include monosomy X and triploidy which account for approximately 20 and 15 of all abnormalities respectively These data are represented in Figure 1 below 2 Autosomal Trisomy Figure 1 Showing the chromosomal findings in products of conception with 46N representing normal results AN6XYB1EN 001 Jan 2015 Page 2 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Principles of the procedure The method employed by Elucigene QST R kits uses the QF PCR Quantitative Fluorescence Polymera
32. roducts are available at www elucigene com AN6XYB1EN 001 Jan 2015 Page 36 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use References Goddijn M Leschot NJ Genetic aspects of miscarriage Baillieres Best Pract Res Clin Obstet Gynaecol 2000 Oct 14 5 855 65 Gardner RJM Sutherland GR 2004 Chromosome Abnormalities and Genetic Counselling New York Oxford University Press 339 360 Mansfield E S Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms Human Molecular Genetics 1993 2 1 43 50 Mann K Fox SP Abbs SJ Yau SC Scriven PN Docherty Z Mackie Ogilvie C Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis The Lancet 2001 358 9287 1057 1061 Mann K Donaghue C Fox SP Docherty Z Mackie Ogilvie C Strategies for the rapid prenatal diagnosis of chromosome aneuploidy European Journal of Human Genetics 2004 12 907 915 Mackie Ogilvie C Donaghue C Fox SP Docherty Z Mann K Rapid prenatal diagnosis of an aneuploidy using Quantitative Fluorescence PCR QF PCR Journal of Histochemistry and Cytochemistry 2005 53 3 285 288 Deutsch S Choudhury U Merla G Howald C Sylvan A Antonarakis SE Detection of aneuploidies by paralogous sequence quantification Journal of Medical Genetics 2004 41 908 915
33. s determines the lowest point in the analysable range Ensure the maximum analysis range encompasses the largest peak of the size standard e g 500bp of GS500LIZ or 600bp of GS600LIZv2 Input the new values into the QST R Analysis file accessing it as described above Figure 6 Finding the minimum range using sample raw data ONE E e m0 AN6XYB1EN 001 Jan 2015 Page 17 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Analysis of Imported QST R PL Files 1 In the main GeneMapper window select the QST R Table Settings v02 Figure 7 Figure 7 Setting QST R table settings bu Table Setting QSTR Table Settings v02 m 2 Under Analysis Method select QSTR Analysis v02 Fill down each column by pressing Ctrl D Repeat this process selecting QSTR PL for under the Panel heading and QSTR under the Size Standard heading Each time remember to fill down by pressing Ctrl D to ensure each setting is applied to the full list of samples 3 Click to initiate sample analysis Assign a project name when prompted Reviewing QST R Data 1 Select the sample for analysis highlight sample row 2 Click n to Display Plots 3 Select the QST R Plot settings Figure 8 Figure 8 QST R Plot settings drop down menu Samples Plot File Edit View Tools Alleles Help p Plot Setting QSTR Plot Settings Y E j fu Li
34. se Chain Reaction technique 3 6 Using PCR amplification fluorescent dye labelled primers target highly polymorphic regions of DNA sequence called short tandem repeats STRs that are located on the chromosomes of interest Each targeted STR marker is specific to the chromosome on which it is located thus the copy number of the STR marker can be diagnostic of the copy number of the chromosome Informative STR markers have been selected that exhibit a high heterogeneity so that copy number can be easily determined A normal diploid sample has the normal complement of two of each of the somatic chromosomes thus two alleles of a chromosome specific STR are determined by the QF PCR technique as two peaks in a 1 1 ratio The observation of an extra STR allele as either a three peak pattern in a 1 1 1 ratio or two peak pattern in a 2 1 or 1 2 peak ratio is diagnostic of the presence of an additional sequence which in turn may represent an additional chromosome as in the case of a trisomy Amplified products of the QF PCR technique are analysed quantitatively on a capillary electrophoresis Genetic Analyzer to determine the copy number of the analysed STR markers Warnings and Precautions 1 The normal DNA Control provided in the kits has been independently tested and found to be negative for Hepatitis B Virus HBV Hepatitis C Virus HCV and Human Immunodeficiency Virus HIV 1 and 2 2 Care should be taken when handling material of human origin A
35. shown as seen in Figure 11 click Yes in order to proceed Figure 11 Allowing security access Security Warning A Do you want to make this file a Trusted Document This file is on a network location Other users who have access to this network location may be able to tamper with this file What 5 the risk E Do not ask me again for network files 4 Paste the data copied from the GeneMapper data table see above Copying Data Table using Ctrl V into the top left cell in the outlined area See Figure 12 AN6XYB1EN 001 Jan 2015 Page 20 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Figure 12 Importing raw GeneMapper data into the QST R PL Report Template gt Pancet Sample ID Elucigen Run Co ord ISTR PL Report Te jl ate Filename Software GeneMapper b RED OUTLINED CELL BELOW Area 1 Area2 Area3 A1 A2 Warning 1 Check 2 Check 3 Check Comment D135305 Absent 0138325 Absent 0135634 Absent 01358628 Absent D155827 Absent D155659 Absent FESFPS Ab 0158151 Absent D165753 Absent D1652624 Absent 01652621 Absent 0165539 Ab 01851002 Absent 0185819 Absent 0185535 Absent 0185386 Absent D21511 Absent 02151437 Absent 02151442 Absent 02151411 Absent 0225686 Absent 0225685 Absent 0225689 Absent 0225683 Absent AMEL Absent TAF9 Absent SRY Absent CONCLUSION NT DATA CHECKED DATE 5 The calculated ratios for each marker will now be shown in the data table
36. ss Assay Instructions for Use Limitations to the Procedure This test is designed to detect specific chromosomal trisomies and sex chromosome aneuploidies as detailed in the Instructions for Use It may not detect structural rearrangements involving the chromosomes tested and will not detect abnormalities in any other chromosomes Mosaicism for the chromosomes tested may not be detected A QST R PL result can only be directly applied to the tissue tested and may not represent the fetal karyotype Maternal cell contamination MCC and confined placental mosaicism CPM may result in discrepancies between the QST R PL and karyotype results Note Heterozygosities of the markers used were derived from a random set of samples submitted for routine analysis from a predominantly Northern European Caucasian population Any calculations using these heterozygosities strictly only apply to the population from which the samples were taken A small study using locally derived samples may be carried out as part of a validation study to establish heterozygosities in the population to be tested It is not expected that population variation will significantly alter the overall informativeness of the assay Disclaimer Hesults from this diagnostic assay should be interpreted in conjunction with other laboratory and clinical data available to the clinician These Elucigene reagents are supplied for n Vitro diagnostic testing Further details of Elucigene QST R p
37. t the reaction mix be dispensed into 0 2ml PCR vials in 10ul volumes and frozen at 20 C Ensure that vial contents are thoroughly thawed and mixed before dispensing The Control DNA should be frozen at 20 C Materials required but not provided General Laboratory consumables gloves screw capped microfuge tubes 0 2ml PCR vials or microtitre plates recommended by the manufacturer of the thermal cycler used pipette tips Laboratory equipment precision pipettes 2 sets 1 for pre amplification and 1 for post amplification handling preferably positive displacement pipettes protective clothing vortex mixer microfuge 96 well microtitre plate centrifuge PCR Amplification Thermal cycler to accommodate 96 well microtitre plates or 0 2ml vials with a temperature accuracy of 1 C between 33 C and 100 C and static temperature uniformity of 1 C QST R PL has been validated and shown to perform to specification on the following recommended Thermal Cycler platforms Life Technologies GeneAmp 9700 Life Technologies Veriti Dx Running in Standard mode Life Technologies Veriti Dx Running in 9700 simulation mode Life Technologies Proflex Running in Standard mode Life Technologies Proflex Running in 9700 simulation mode Note Peak signal intensities may increase when using the Veriti and Proflex thermal cycler platforms compared to the GeneAmp 9700 platform AN6XYB1EN 001 Jan 2015 Page 5 of 37 Elucigene amp QST R
38. tained with input DNA range of 1 25ng to 10ng Note sample injection settings can be modified to suit the amount of amplicon produced during the PCR reaction which can vary due to amount of input genomic DNA added Less amplicon can be applied to the column for analysis by reducing time of injection Conversely more amplicon can be applied to the column for analysis by increasing either time or voltage of injection Previously amplified samples can be re injected multiple times for re analysis AN6XYB1EN 001 Jan 2015 Page 6 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Test Protocol Amplification Procedure Note to minimise the risk of contamination steps 3 5 must be carried out in an area free from DNA Steps should also be taken to avoid contamination with PCR product 1 Program the thermal cycler for a single step cycle to activate the DNA polymerase at 95 C for 15 minutes linked to an amplification cycling program of 30 seconds at 95 C denaturation 1 minute and 30 seconds at 59 C annealing and 1 minute and 30 seconds at 72 C extension for 26 cycles This should be linked to a 30 minutes time delay file at 72 C extension on the final cycle Enzyme Activation Cycling Final Extension 15 min 30 secs Ambient 1 min 30 secs Temperature T 2B Cycles A negative water control must be included in each PCR run It may also be considered appropriate to include other con
39. the Data Processing box when analysis is complete Figure 20 Data Processing Box Events Checking options Processing Samples C11 Run AB3130A 2014 10 24 15 12 0299 2014 10 24 fsa Completed Checking Calibration 1 samples processed Analysis Time 0 14s AN6XYB1EN 001 Jan 2015 Page 28 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Main Analysis Window The Main Analysis window Figure 21 of GeneMarker has an easy to use layout This layout includes The sample files list displayed on the left side of the window The Synthetic Gel Image displayed at the top of the window Data Electropherograms below the gel image A Report Table displayed on the right side of the window In this window it is important to check that all the appropriate peaks in each profile have been called correctly 1 Double click on each sample in turn in the sample file tree on the left hand side of the screen Right click on any peaks in question and choose from the options in the dialogue box e g edit or delete allele confirm or unconfirm as appropriate 2 From the Main Analysis window select the Applications drop down menu option from the top of the screen Select Trisomy Analysis The Trisomy Analysis Settings box will then open Figure 22 Figure 21 Main Analysis Window Sample File Tree Synthetic Gel Image Report Table Electropherogram Mo adl EL
40. the longer length allele or where A1 A2 will give a result in the region of 0 45 to 0 65 when the peak representing the shorter length allele is smaller in area than the peak representing the longer length allele 7 2 Three peaks of comparable height present The ratio of the peaks will be classed as 1 1 1 and their values fall within the normal range of 0 8 1 4 although for alleles separated by more than 24bp an allele ratio of up to 1 5 is acceptable If this does not occur then it may be due to one of the factors mentioned in step 6 To interpret a result as normal at least two informative markers consistent with a di allelic genotype are required with all other markers being uninformative A normal result indicates the normal complement of two for the chromosome tested Peak area ratios that fall between the normal and abnormal ranges are classed as inconclusive Inconclusive results may be resolved by using the single chromosome kits If both normal and abnormal allele patterns are obtained for a single chromosome then it is recommended that follow up studies are carried out to identify the reason for the discrepant results prior to any conclusions being reached In rare cases allele size ranges for markers may overlap If this is suspected analysis with the single chromosome kits may resolve this Analysis of Sex chromosome markers AMEL TAF9 and SRY The AMEL marker amplifies non polymorphic sequences on the X 104bp and Y 1
41. tors such as Whole chromosome trisomy Partial chromosome trisomy including sub microscopic duplications Mosaicism Contaminating second genotype e g maternal twin external otutters causing skewing Preferential amplification of one allele causing skewing Primer site polymorphisms somatic microsatellite mutations AN6XYB1EN 001 Jan 2015 Page 10 of 37 10 11 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use The Guide to Interpretation gives examples of typical profiles for many of these Homozygous markers are uninformative since a ratio cannot be determined To interpret a result as abnormal i e trisomy present at least two informative markers consistent with a tri allelic genotype are required with all other markers being uninformative It is not recommended to interpret a result as abnormal based on information from only one marker If required follow up testing with the single chromosome kits i e Elucigene QST R 13 Elucigene QST R 18 Elucigene QST R 21 may provide sufficient information for interpretation Trisomy is determined by either 7 1 Two peaks of uneven height due to one of the peaks representing two alleles which are common to one or both parents In this case the ratio between the two peaks will be classed as 2 1 or 1 2 such that A1 A2 will give a result in the region of 1 8 to 2 4 when the peak representing the shorter length allele is greater in area than the peak representing
42. trols e g positive normal DNA control supplied and positive trisomy control DNA not supplied Thaw sufficient vials of pre aliquoted QST R PL reaction mix for the number of samples and controls to be run see note under Materials Provided and centrifuge the vials at 12 000g for 10 seconds Using separate pipette tips add 2 5ul of test DNA to a sample vial containing 10ul QST R PL reaction mix and mix by pipetting up and down Do this for all samples to be tested Do not add DNA to the PCR vial for the negative control instead add 2 5ul of sterile distilled water Briefly centrifuge the vials until all liquid is at the bottom of each vial Place all vials firmly in the thermal cycler block Initiate the 95 C activation program followed by the amplification program see step 1 On completion of the amplification program the samples may be stored at room temperature overnight or at 2 8 C for up to 7 days before analysis by capillary electrophoresis AN6XYB1EN 001 Jan 2015 Page 7 of 37 Elucigene amp QST R PL Pregnancy Loss Assay Instructions for Use Capillary Electrophoresis It is recommended that each user ensure that the chosen equipment is used according to the manufacturer s instructions and is compatible with this test In this context the key parameters are the polymer and the capillary array Optimal results can be obtained using the following capillary electrophoresis conditions on an ABI3130 or ABI3500 Genetic Analyzer
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