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Yfiler® Plus PCR Amplification Kit User Guide

1. 3130xl 3500 3500xL Allele Standard Standard Standard mean deviation eae deviation Mean deviation 15 97 28 97 34 0 024 0 037 97 30 97 35 0 027 0 038 97 23 97 24 0 021 0 035 16 101 47 101 53 0 022 0 031 101 52 101 55 0 010 0 033 101 42 101 43 0 034 0 040 17 105 65 105 72 0 021 0 030 105 73 105 75 0 025 0 038 105 62 105 64 0 023 0 035 18 109 81 109 88 0 022 0 037 109 89 109 92 0 023 0 056 109 77 109 80 0 027 0 038 19 113 92 113 98 0 029 0 032 114 01 114 04 0 028 0 060 113 89 113 91 0 032 0 038 20 117 90 117 95 0 022 0 035 118 01 118 02 0 026 0 044 117 88 117 88 0 022 0 029 21 121 86 121 93 0 022 0 036 121 97 122 01 0 040 0 044 121 84 121 86 0 036 0 042 22 125 85 125 93 0 027 0 033 125 98 126 00 0 032 0 048 125 85 125 87 0 032 0 038 23 129 87 129 94 0 029 0 033 130 00 130 04 0 042 0 058 129 88 129 89 0 032 0 040 24 133 89 133 96 0 026 0 033 134 04 134 05 0 030 0 041 133 92 133 94 0 024 0 044 DYS458 11 119 84 119 98 0 027 0 034 120 25 120 28 0 039 0 065 120 11 120 13 0 039 0 041 12 123 66 123 81 0 026 0 038 124 10 124 13 0 042 0 056 123 96 123 98 0 026 0 040 13 127 52 127 66 0 025 0 041 127 97 128 01 0 035 0 053 127 82 127 85 0 027 0 040
2. ekla nedi Alleles included in Yfiler Plus Allelic Ladder a DYS576 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 6 FAM 19 DYS3891 9 10 11 12 13 14 15 16 17 13 DYS635 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 24 DYS3891I 24 25 26 27 28 29 30 31 32 33 34 35 29 DYS627 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 21 DYS460 7 8 9 10 11 12 13 14 VIC 11 DYS458 11 12 13 14 15 16 17 18 19 20 21 22 23 24 17 DYS19 9 10 11 12 13 14 15 16 17 18 19 15 YGATAH4 8 9 10 11 12 13 14 15 13 DYS448 14 15 16 17 18 19 20 21 22 23 24 19 DYS391 5 6 7 8 9 10 11 12 13 14 15 16 11 DYS456 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 NED 15 DYS390 17 18 19 20 21 22 23 24 25 26 27 28 29 24 DYS438 6 7 8 9 10 11 12 13 14 15 16 12 DYS392 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 13 DYS518 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 37 DYS570 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 TAZ 17 DYS437 10 11 12 13 14 15 16 17 18 15 DYS385 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 ie DYS449 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 30 DYS393 7 8 9 10 11 12 13 14 15 16 17 18 SID 13 DYS439 6 7 8 9 10
3. 5 Request the software license file by performing steps 1a 1b and 1c as listed on the activation screen The license file will be emailed to you 6 Obtain the software license file from your email 7 Make a copy of the software license file and keep in a safe location 8 Copy the software license file to the desktop of the Data Collection Software v4 computer 9 If the Software Activation dialog box has closed select Tools License Manager 10 Click Browse then navigate to the software license file saved on your computer 11 Click Install and Validate License A message is displayed when the license is installed and validated 12 Click Close 42 Yfiler Plus PCR Amplification Kit User Guide Section 3 2 3130 3130xl instruments Prepare samples for electrophoresis on the 3130 3130xl instruments Perform spectral Perform a spectral calibration using the DS 36 Matrix Standard J6 Dye Set calibration Part no 4425042 The following figure is an example of a passing 6 dye spectral calibration 50 100 150 200 250 Intensity vs Pixel Number 2000 3000 Intensity vs Scan Number Prepare samples for electrophoresis on the 3130 3130x instruments Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per reaction GeneScan 600 LIZ Size Standard v2 0 0 4 uL
4. Parameter Setting Size Standard GS600_LIZ 60 460 Size Range Partial Sizing Start Size 60 bp Sizing Stop Size 460 bp Size Calling Method Local Southern Method After checking the Use Baselining box Baseline Window Pts 33 Peak Window Size 13 c Click Save 3 Add the QC protocol to the HID assay Yfiler Plus PCR Amplification Kit User Guide 37 Perform electrophoresis Prepare samples for electrophoresis on the 3500 3500xL instruments Perform spectral Perform a spectral calibration using the DS 36 Matrix Standard J6 Dye Set calibration Part no 4425042 The following figure is an example of a passing 6 dye spectral calibration gt Intemety ve Scan Marker Intensity vs Piet Number Cobbeated Data Hm z m E m m z z m Interne va Scan Number EGER nos Prepare samples for electrophoresis on the 3500 3500xL Prepare the samples for electrophoresis immediately before loading 1 instruments 2 3 4 38 Calculate the volume of Hi Di Formamide and GeneScan 600 LIZ Size Standard v2 0 needed to prepare the samples Reagent Volume per reaction GeneScan 600 LIZ Size Standard v2 0 0 4 uL Hi Di Formamide 9 6 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the ap
5. 59 Examine and edit a project oe 60 FOF moreinformation unne gean tende ern ida Jb aki eee 61 Overview of GeneMapper D X Software v1 4 GeneMapper ID X Software v1 4 or higher analyzes 4 dye 5 dye and 6 dye data and is required to correctly analyze data generated using the Yfiler Plus Kit After electrophoresis the data collection software stores information for each sample in a fsa or hid file Using GeneMapper ID X Software v1 4 or higher enables you to analyze and interpret the data from the fsa or hid files Instruments Refer to Instrument and software overview on page 15 for a list of compatible instruments Before you start When using GeneMapper ID X Software v1 4 or higher to perform human identification HID analysis with Yfiler Plus kits be aware that e HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder e Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subs
6. Find Name Containing a Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Type Description CE_F_HID_G5500 75 400 2007 08 09 13 23 gmidx Advanced CE_F_HID_G5500 75 450 2007 08 09 13 24 gmidx Advanced CE_G5_HID_G5500 j201 1 04 18 13 15 gmidx Advanced 3 Complete the Name field as shown below or with a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the drop down list In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified on page 57 Size Standard Editor Edit Size Standard Description Name G5600_LIZ 60 460 Security Group GeneMapper ID X Security Group v Description Size Standard Dye Orange v Size Standard Table Size in Basepairs s oo d i ej n 58 Yfiler Plus PCR Amplification Kit User Guide Chapter 4 Analyze Data 4 Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper D X Software 1 In the Project window select Edit Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Parameter Settings Sample Type S
7. IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single and multiple capillary runs with larger size variations seen between samples injected in multiple capillary runs We recommend the above frequency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment It is critical to genotype using an allelic ladder run under the same conditions as the samples because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions Yfiler Plus PCR Amplification Kit User Guide 33 et for electrophoresis Section 3 1 3500 3500xL instruments Set up the 3500 3500xL instruments for electrophoresis Reagents and parts Appendix C Ordering information on page 109 lists the required materials not supplied with this kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Do not refreeze kit components after thawing 34 Yfiler Plus PCR Amplification Kit User Guide Electrophoresis software setup and Section 3 1 3500 3500xL instruments Set u
8. More than expected number of alleles present at a locus Secondary gene duplication at DYS385 and or DYF387S1 Presence of exogenous DNA Some samples may exhibit uneven peak height ratios at these markers due to either the stochastic effects of the PCR or a secondary duplication event in one of the alleles We recommend that allele calls be made based on peaks that are present conservative approach unless additional evidence is gathered to conclusively demonstrate that a secondary duplication event has taken place Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Amplification of stutter product 1 repeat unit position Mixed sample Incomplete 3 A base addition n 1 nt position See Stutter products on page 69 Be sure to include the final extension step of 60 C for 22 minutes in the PCR Signal exceeds dynamic range of instrument off scale data Check that you are using the recommended number of PCR cycles see Perform PCR on page 31 Repeat PCR amplification using reduced input DNA amount or use your laboratory s SOP to analyze off scale data Gene duplication To confirm duplication re amplify with a different sample from the same individual Poor spectral separation bad matrix Follow the steps for creating a spectral file Confirm that Filter Set J6 modules are installed and used for analysis Too much DNA in react
9. Data Collection and GeneMapper D X Software Instrument and software compatibility About multicomponent analysis How multicomponent analysis works Instrument and software overview Chapter 1 Overview This section provides information about the Data Collection Software versions required to run the Yfiler Plus PCR Amplification Kit on specific instruments The Data Collection Software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument measures sample fluorescence with its detection system the Data Collection Software collects the data and stores it The Data Collection Software stores information about each sample in a sample file fsa which is then analyzed by the GeneMapper ID X Software Table 2 Software specific to each instrument Instrument Operating Data Collection Analysis software system Software 3500 3500xL Windows XP 3500 Series GeneMapper D X Software e Windows Data Collection v1 4 or higher Vista Software v1 0 and v2 0 3130 3130x Windows 7 4 0 Life Technologies fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis is the process that separates the six different fl
10. 14 131 37 131 53 0 033 0 038 131 84 131 90 0 040 0 069 131 70 131 74 0 024 0 041 15 135 26 135 41 0 020 0 043 135 77 135 80 0 035 0 059 135 62 135 65 0 030 0 038 16 139 17 139 32 0 027 0 046 139 68 139 72 0 041 0 051 139 56 139 58 0 028 0 044 17 143 06 143 24 0 030 0 044 143 61 143 64 0 028 0 049 143 47 143 52 0 030 0 040 18 147 14 147 31 0 030 0 045 147 70 147 72 0 033 0 054 147 56 147 59 0 035 0 047 19 151 22 151 39 0 029 0 050 151 77 151 83 0 040 0 060 151 65 151 68 0 034 0 043 20 155 22 155 39 0 028 0 047 155 79 155 82 0 031 0 062 155 65 155 68 0 029 0 049 21 159 12 159 30 0 028 0 049 159 71 159 73 0 028 0 041 159 57 159 61 0 027 0 042 22 163 07 163 26 0 027 0 050 163 65 163 69 0 039 0 063 163 52 163 55 0 035 0 048 23 166 99 167 18 0 019 0 060 167 58 167 61 0 028 0 059 167 45 167 48 0 030 0 042 24 170 92 171 11 0 028 0 064 171 52 171 54 0 042 0 063 171 38 171 42 0 033 0 044 DYS460 7 79 58 79 61 0 031 0 038 79 58 79 60 0 053 0 060 79 38 79 40 0 023 0 039 8 83 76 83 80 0 021 0 040 83 75 83 78 0 042 0 058 83 58 83 60 0 026 0 037 9 87 96 87 98 0 025 0 037 87 92 87 97 0 027 0 035 87 77 87 79 0 029 0 038 10 92 12 92 15 0 024 0 042 92 10 92 13 0 023 0 043 91 95 91 97 0 031 0 044 11 96 29 96 31 0 028
11. 2 uL of Control DNA 007 blank disc to the positive e For 28 cycles 1 uL of Control DNA 007 control well e For 29 cycles 1 uL of Control DNA 007 Note The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number 2 Calculate the volume of each component needed to prepare the reactions using the table below Reaction component Volume per reaction Master Mix 10 0 uL Primer Set 5 0 uL PCR Low TE buffer 10 0 uL Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT This kit has been optimized for a 25 uL PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles 3 Prepare reagents Thaw the Master Mix and Primer Set then vortex for 3 seconds Centrifuge briefly before opening the tubes or bottles IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore do not require subsequent thawing Do not refreeze the reagents 4 Pipet the required volumes of components into an appropriately sized polypropylene tube 5 Vortex the reaction mix for 3 seconds then centrifuge briefly 6 Dispense 25 uL of the reaction mi
12. 0 037 0 043 34 304 81 304 89 0 024 0 035 304 93 304 96 0 025 0 060 304 85 304 86 0 031 0 038 35 308 89 308 97 0 027 0 041 309 01 309 03 0 036 0 046 308 93 308 94 0 040 0 050 DYS390 17 144 14 144 23 0 026 0 044 144 14 144 19 0 038 0 042 144 09 144 11 0 029 0 039 18 148 04 148 12 0 020 0 038 148 06 148 10 0 021 0 051 147 98 148 01 0 035 0 043 19 151 96 152 04 0 026 0 037 151 99 152 02 0 026 0 043 151 92 151 93 0 029 0 039 20 156 15 156 25 0 027 0 046 156 16 156 20 0 026 0 041 156 09 156 12 0 025 0 038 21 160 16 160 24 0 031 0 039 160 17 160 18 0 000 0 043 160 09 160 10 0 025 0 037 22 164 21 164 30 0 024 0 039 164 21 164 24 0 020 0 061 164 15 164 17 0 030 0 041 23 168 34 168 42 0 026 0 039 168 31 168 34 0 030 0 048 168 25 168 28 0 032 0 044 24 172 34 172 42 0 024 0 038 172 30 172 32 0 032 0 055 172 25 172 27 0 025 0 033 25 176 33 176 41 0 024 0 034 176 30 176 33 0 029 0 044 176 24 176 26 0 027 0 037 26 180 35 180 44 0 023 0 036 180 33 180 34 0 032 0 041 180 27 180 29 0 026 0 041 27 184 33 184 42 0 019 0 030 184 32 184 34 0 040 0 052 184 26 184 28 0 031 0 044 28 188 44 188 53 0 026 0 041 188 41 188 43 0 032 0 050 188 36 188 38 0 029 0 043 29 192 49 192 58 0 027 0 042 192 46 192 48 0 022
13. 0 038 114 32 114 34 0 017 0 037 15 118 29 118 32 0 021 0 030 118 32 118 34 0 022 0 037 118 23 118 25 0 035 0 038 16 122 21 122 24 0 023 0 041 122 23 122 26 0 032 0 041 122 15 122 17 0 027 0 036 17 126 18 126 21 0 026 0 041 126 21 126 22 0 030 0 042 126 13 126 14 0 027 0 042 18 130 16 130 19 0 021 0 041 130 18 130 20 0 015 0 039 130 11 130 12 0 028 0 034 19 134 15 134 19 0 027 0 038 134 18 134 21 0 025 0 038 134 11 134 13 0 026 0 041 20 138 19 138 23 0 026 0 043 138 20 138 25 0 027 0 046 138 14 138 16 0 027 0 036 21 142 27 142 30 0 024 0 033 142 29 142 31 0 024 0 037 142 23 142 25 0 034 0 040 22 146 38 146 42 0 021 0 028 146 39 146 42 0 020 0 033 146 34 146 35 0 028 0 038 23 150 50 150 55 0 027 0 031 150 49 150 51 0 035 0 046 150 45 150 47 0 025 0 039 24 154 62 154 67 0 023 0 040 154 61 154 64 0 027 0 039 154 57 154 60 0 027 0 036 25 158 75 158 81 0 024 0 038 158 73 158 75 0 034 0 038 158 69 158 70 0 016 0 029 26 162 84 162 88 0 027 0 042 162 80 162 82 0 019 0 042 162 76 162 79 0 031 0 040 DYS576 10 72 26 72 37 0 028 0 050 72 79 72 82 0 041 0 073 72 45 72 47 0 038 0 045 11 76 42 76 55 0 027 0 043 77 02 77 03 0 059 0 066 76 69 76 71 0 028 0 047 12 80 56 80 68 0 020 0 038 81 18 81 20 0 039 0 064 80 88 80 89 0
14. Hi Di Formamide 9 6 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your experiments and results 2 Pipet the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly Yfiler Plus PCR Amplification Kit User Guide 43 Prepare samples for electrophoresis on the 3130 3130xl instruments 4 Into each well of a MicroAmp Optical 96 Well Reaction Plate add e 10 uL of the formamide size standard mixture e 1 ul of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di Formamide 5 Seal the reaction plate with appropriate septa then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Place the plate assembly on the autosampler yon KO Ee ON Start the electrophoresis run 44 Yfiler Plus PCR Amplification Kit User Guide Analyze Data Overview of GeneMapper ID X Software v1 4 an oan onee 45 Set up GeneMapper ID X Software for data analysis 47 Analyze and edit sample files with GeneMapper ID X Software
15. Plus PCR Amplification Kit User Guide 79 80 Experiments and Results Extra Peaks in the electropherogram Figure 20 Examples of reproducible artifacts in data produced on the Applied Biosystems 3500 3500xL The top panel is TAZ412 the middle panel is FAM348 and the bottom panel is Y391 n 10 wan pincode J Dt gJ 360 380 1000 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Characterization of loci Characterization of loci SWGDAM Guideline 3 1 Nature of the polymorphisms Inheritance Mapping The basic characteristics of a genetic marker must be determined and documented SWGDAM December 2012 This section describes basic characteristics of the 27 loci that are amplified with the Yfiler Plus Kit These loci have been extensively characterized by other laboratories Gusmao et al 1999 Butler et al 2002 Gonzalez Neira et al 2001 Hall and Ballantyne 2003 Redd et al 2002 Schoske et al 2004 Ballantyne et al 2012 Ballantyne et al 2014 DYS392 and DYS481 are trinucleotide repeats DYS438 is a pentanucleotide repeat and DYS448 is a hexanucleotide repeat Their allele differences result from differences in the number of repeat units 3 bp 5 bp and 6 bp respectively The remaining Yfiler Plus Kit loci are tetranucleotide short tandem repeat STR loci The length differences among alleles of these partic
16. Whatman and FTA are registered trademarks of Whatman Limited NUCLEIC CARD is a trademark and Copan is a registered trademark of Copan Italia S P A and used by Life Technologies under their permission Harris Micro Punch is a registered trademark of Harris Joel S TA Shunderson Communications CPA200 and CPA300 are trademarks of Newlab Engineering S r l VWR Scientific is a registered trademark of VWR International Inc Robbins Scientific is a registered trademark of Molecular Bioproducts Inc Agilent is a registered trademark of Agilent Technologies Inc Adobe Acrobat and Reader are registered trademarks of Adobe Systems Incorporated Contents About This Guide semen en kk kk kk kk kk KK KK kk kk kk kk kk kk kok kok kk Sle 7 Revision HIStory oere se ene eee ade ee er ade er ee Py_iyiyi 7 PURPOSE reren dB A a BRE Wd RR Aard JJI JJ N nd ee ZM JJAJAMID DDDM AMN 7 M CHAPTER 1 Overview kk kk kk kk kk kk kk kk eeen 9 PrOd cEOVERVIEW s tit ansich kla lar terende el yeh etende la NA EK Wek e ded y k 9 gill e elo SE Mene ev ED ae A EA DE EEES r ee 9 Product description ss kad eren ier eer Pet ki ben a el aE ans Bl 9 Stelle ahd pha ade tach ee nae a eee wie rra 9 About the primers sass raba manman penance ve eddie pe she diene foe Remedies Ree AS een 10 Lociamplified by the Kit ai AX kek pokes ree kl eel ene Golan ed 10 Allelic ladder profile neee kk kK kk kk kk kk kk kk kk kk kk kk kk kk k 11 Control DNA 00Zpr
17. and that met reproducible performance standards when using a peak amplitude threshold of 175 RFUs It is our opinion that while these experiments are not exhaustive they are appropriate for a manufacturer of STR kits intended for forensic and or parentage testing use Yfiler Plus PCR Amplification Kit User Guide 63 5 Experiments and Results Developmental validation Developmental validation SWGDAM guideline 2 2 1 SWGDAM guideline 3 9 2 PCR components 64 Developmental validation is the acquisition of test data and determination of conditions and limitations of a new or novel DNA methodology for use on forensic database known or casework reference samples SWGDAM December 2012 The reaction conditions needed to provide the required degree of specificity and robustness should be determined These include but are not limited to thermal cycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM December 2012 We examined the concentration of each component of the Yfiler Plus Kit and established that the concentration of each component was within the range where data indicated that the amplification met the required performance criteria for specificity sensitivity and reproducibility For example 1 ng of DNA Control 007 was amplified in the presence of varying concentrations of magnesium chloride and the results were analyzed on an Applied B
18. oe 257 0 24 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 DY5576 DYS389II Blue 314 2 29 24 25 26 27 28 29 30 31 32 33 34 35 DYS3891 DYS627 Blue 393 5 21 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 DYS635 DYS460 Green 113 1 11 7 8 9 10 11 12 13 14 DYS3891II DYS458 Green 177 0 17 11 12 13 14 15 16 17 18 19 20 21 22 23 24 DYS627 DYS19 Green 229 5 15 9 10 11 12 13 14 15 16 17 18 19 DYS460 GATA_H4 Green 269 4 13 8 9 10 11 12 13 14 15 DYS458 DYS448 Green 345 2 19 14 15 16 17 18 19 20 21 22 23 24 DYS19 DYS391 Green 402 5 11 5 6 7 8 9 10 11 12 13 14 15 16 GATA_H4 DYS456 Yellow 135 0 15 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 DYS448 DYS390 Yellow 197 2 24 17 18 19 20 21 22 23 24 25 26 27 28 29 DYS391 DYS438 Yellow 263 5 12 6 7 8 9 10 11 12 13 14 15 16 DYS456 DYS392 Yellow 326 8 13 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 DYS390 DY5518 Yellow 406 0 37 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 DYS438 DY5570 Red 167 5 17 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 DYS392 DYS437 Red 215 3 15 10 11 12 13 14 15 16 17 18 DYS518 DYS385 Red 318 4 11 14 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2
19. 0 047 192 41 192 44 0 034 0 042 DYS391 5 352 78 352 85 0 024 0 035 353 42 353 45 0 038 0 057 353 34 353 36 0 034 0 049 6 356 84 356 91 0 032 0 042 357 46 357 51 0 032 0 054 357 39 357 42 0 036 0 051 74 360 69 360 78 0 030 0 036 361 40 361 43 0 026 0 057 361 32 361 35 0 040 0 049 8 364 75 364 85 0 034 0 045 365 47 365 49 0 019 0 035 365 40 365 43 0 033 0 057 9 368 74 368 85 0 023 0 042 369 47 369 50 0 024 0 060 369 39 369 43 0 033 0 046 10 372 75 372 87 0 020 0 040 373 47 373 48 0 036 0 076 373 40 373 44 0 032 0 050 11 376 75 376 87 0 029 0 044 377 47 377 47 0 019 0 047 377 38 377 42 0 037 0 044 12 380 81 380 90 0 031 0 052 381 44 381 48 0 018 0 050 381 36 381 39 0 028 0 049 Yfiler Plus PCR Amplification Kit User Guide 95 Table of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard Mean deviation mean deviation mean deviation 13 384 93 385 02 0 029 0 045 385 56 385 60 0 020 0 063 385 47 385 50 0 037 0 042 14 388 98 389 07 0 024 0 038 389 60 389 62 0 023 0 050 389 50 389 54 0 028 0 036 15 393 02 393 08 0 026 0 046 393 63 393 67 0 024 0 043 393 55 393 57 0 033 0 045 16 397 05 397 12 0
20. 025 175 37 175 40 0 020 0 046 175 23 175 24 0 026 0 039 17 179 23 179 28 0 013 0 034 179 40 179 41 0 005 0 032 179 25 179 27 0 041 0 045 94 Yfiler Plus PCR Amplification Kit User Guide Appendix ATable of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard mean deviation Mean deviation mean deviation DYS389II 24 265 02 265 11 0 025 0 032 265 16 265 20 0 023 0 060 265 12 265 12 0 030 0 044 25 269 11 269 20 0 024 0 040 269 23 269 25 0 030 0 052 269 17 269 18 0 030 0 042 26 273 08 273 17 0 021 0 043 273 23 273 24 0 026 0 040 273 15 273 17 0 028 0 042 27 277 22 277 32 0 024 0 039 277 36 277 39 0 036 0 054 277 29 277 31 0 030 0 042 28 281 15 281 25 0 020 0 048 281 29 281 32 0 032 0 047 281 21 281 22 0 034 0 043 29 285 01 285 10 0 019 0 041 285 14 285 16 0 029 0 043 285 07 285 09 0 028 0 041 30 289 17 289 26 0 026 0 047 289 31 289 33 0 027 0 039 289 23 289 25 0 034 0 039 31 293 15 293 24 0 023 0 043 293 27 293 31 0 037 0 040 293 19 293 21 0 023 0 037 32 297 02 297 11 0 027 0 035 297 14 297 15 0 029 0 059 297 04 297 07 0 028 0 044 33 300 97 301 06 0 025 0 041 301 10 301 13 0 039 0 049 301 02 301 03
21. 031 0 042 397 66 397 70 0 022 0 057 397 58 397 61 0 032 0 047 DYS392 4 273 67 273 77 0 028 0 036 274 35 274 38 0 045 0 056 274 25 274 28 0 039 0 047 5 276 63 276 74 0 027 0 044 277 36 277 39 0 048 0 063 277 25 277 28 0 030 0 048 6 279 61 279 73 0 021 0 038 280 32 280 35 0 033 0 051 280 22 280 25 0 045 0 054 7 282 64 282 75 0 023 0 032 283 35 283 39 0 042 0 056 283 26 283 29 0 037 0 048 8 285 54 285 66 0 026 0 030 286 23 286 25 0 050 0 065 286 12 286 16 0 032 0 048 9 288 52 288 60 0 027 0 036 289 12 289 14 0 040 0 057 289 00 289 04 0 034 0 044 10 291 25 291 38 0 026 0 040 291 97 292 00 0 049 0 065 291 87 291 89 0 034 0 052 11 294 30 294 42 0 026 0 039 295 04 295 05 0 051 0 072 294 91 294 94 0 030 0 055 12 297 26 297 38 0 026 0 038 297 96 297 99 0 036 0 058 297 84 297 88 0 035 0 058 13 300 19 300 30 0 031 0 045 300 89 300 90 0 037 0 067 300 77 300 80 0 038 0 047 14 303 01 303 13 0 027 0 036 303 73 303 74 0 048 0 061 303 59 303 63 0 036 0 050 15 306 00 306 12 0 028 0 040 306 70 306 74 0 043 0 070 306 59 306 63 0 033 0 052 16 309 03 309 15 0 026 0 044 309 71 309 73 0 041 0 073 309 58 309 62 0 038 0 053 17 311 93 312 06 0 027 0 044 312 67 312 70 0 027 0 064 312 54 312 58 0 027 0 047 18 31
22. 050 20 313 18 313 29 0 038 0 048 313 87 313 91 0 051 0 079 313 86 313 90 0 040 0 060 21 319 33 319 45 0 036 0 049 320 06 320 09 0 035 0 052 320 06 320 10 0 039 0 066 22 325 50 325 63 0 041 0 053 326 21 326 23 0 038 0 062 326 22 326 26 0 049 0 060 23 331 46 331 61 0 036 0 056 332 16 332 19 0 039 0 070 332 16 332 20 0 045 0 066 24 337 39 337 53 0 038 0 051 338 10 338 14 0 024 0 077 338 11 338 15 0 040 0 060 DYS449 22 325 50 325 57 0 027 0 062 325 58 325 61 0 037 0 049 325 63 325 67 0 035 0 049 23 329 59 329 64 0 027 0 064 329 65 329 67 0 028 0 054 329 71 329 73 0 039 0 060 24 333 63 333 66 0 025 0 062 333 66 333 72 0 025 0 041 333 75 333 76 0 032 0 045 25 337 66 337 69 0 031 0 058 337 68 337 72 0 033 0 045 337 76 337 78 0 027 0 040 26 341 69 341 75 0 029 0 052 341 72 341 77 0 025 0 042 341 79 341 81 0 039 0 051 27 345 76 345 87 0 029 0 050 345 80 345 84 0 031 0 049 345 87 345 88 0 032 0 043 28 349 82 349 93 0 028 0 061 349 87 349 89 0 018 0 051 349 93 349 94 0 029 0 039 29 353 87 353 98 0 025 0 053 353 92 353 95 0 020 0 060 353 98 353 99 0 034 0 044 30 357 92 358 03 0 028 0 060 357 97 358 00 0 027 0 042 358 03 358 06 0 035 0 050 31 361 97 362 04 0 036 0 045 362 01 362 04 0 021 0 040 362 07 362 09
23. 054 39 360 82 360 88 0 025 0 044 360 73 360 77 0 036 0 049 360 77 360 79 0 035 0 058 40 364 83 364 91 0 021 0 043 364 76 364 78 0 026 0 046 364 80 364 82 0 030 0 041 41 368 87 368 92 0 021 0 036 368 78 368 80 0 032 0 059 368 82 368 84 0 035 0 046 42 372 87 372 94 0 030 0 038 372 78 372 83 0 033 0 043 372 83 372 85 0 030 0 041 43 376 91 376 98 0 026 0 043 376 81 376 83 0 026 0 057 376 84 376 88 0 038 0 053 44 380 95 381 01 0 024 0 034 380 83 380 86 0 041 0 052 380 88 380 89 0 030 0 049 45 385 00 385 07 0 024 0 038 384 89 384 92 0 019 0 045 384 93 384 95 0 029 0 046 46 389 05 389 14 0 027 0 035 388 96 388 97 0 026 0 054 388 99 389 00 0 038 0 048 47 393 09 393 19 0 023 0 043 393 00 393 02 0 033 0 049 393 04 393 06 0 025 0 053 48 397 12 397 24 0 034 0 047 397 04 397 08 0 028 0 054 397 08 397 12 0 031 0 052 49 401 16 401 26 0 025 0 039 401 08 401 10 0 018 0 041 401 10 401 13 0 031 0 051 DYS533 7 338 37 338 42 0 028 0 035 338 61 338 63 0 036 0 049 338 55 338 56 0 020 0 036 100 Yfiler Plus PCR Amplification Kit User Guide Appendix ATable of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard me
24. 17 18 19 20 21 22 23 24 25 26 27 28 29 DYS19 DYS385 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra Peaks in the electropherogram Figure 12 Minus stutter percentages for the DYS3891 DYS38911 DYS390 and DYS391 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 Percent Stutter 8 9 10 111213 14 15 16 17 18 DYS3891 Yfiler Plus PCR Amplification Kit User Guide 23 24 25 26 27 28 29 30 31 32 33 34 35 36 DYS389ll 16 17 18 19 20 21 22 23 24 25 26 27 282930 4 5 6 7 8 DYS390 9 10 1112 13 14 15 16 17 DYS391 73 74 Experiments and Results Extra Peaks in the electropherogram Figure 13 Minus stutter percentages for the DYS392 DYS393 DYS437 and DYS438 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 2 15 0 14 0 13 0 12 0 11 0 tale 10 0 Percent Stutter 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 DYS392 T 3 4 5 6 7 8 9 1011121314151617 18 19 20 21 6 7 8 9 1011121314 15 1
25. 3 8 oon kk kk kk KK KK KK kk kk kk kk kk kk kk kk kk kk kk kk kk kk 87 Male female mixture studies kk kk kk kk kk kk cece kk kk kk kK KK KK KK KK KK KK KK 87 Yfiler Plus PCR Amplification Kit User Guide 5 Contents Male male mixture studies WWW kk kk kk kk kk kk kk kk kk kK kk kK KK KK KK KK KK KK KK KK KK KK 88 Population dat ner mea W nak nape ede ver etn ke deka vie ed elk Kies enne 89 SWGDAM YSTR Guideline 10 1 nonnen KK KK KK KK KK KK KK KK KK KK KK kk kk 89 OVERVIEW EE 89 Population samples used in these studies oneens een 89 Gene diversity values kk kk kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk eee 89 Analyzing the population data MM kk kk kk kk kk kk kK kk kk kk kK KK KK KK KK KK KK kK kK kk kk KK kK kk 90 Mutalan rale a asana IZZJJJJJMIMMMIEIMNMMNMLAMDDDMDMDIDMIAMDMDMDDMDDJDDMJDJDJDJDJDJDDDMDJD J_ P po02mUM JkM 91 APPENDIX A Table of Precision Results 93 Table of typical precision results kk kk kk kK kk KK kK KK KK KK KK KK KK KK kk kk kk kK kk kk 93 APPENDIX B Troubleshooting ven RR KK KK KK 105 APPENDIX Ordering information c0cceeeeeeees 109 Equipment and materials not included eeen 109 APPENDIX D PCR Work Areas 0 0 0 KK KK KK KK KK KK KK 113 Work area setup and lab design kk kk kk kk kk kk kk kK kK kK kk kK KK KK KK kK kK kK kk kk kk kk kk kk kk 113 PCR setup work area kk kk kk kk kk kk kk k
26. 33 0 038 0 055 264 90 264 93 0 045 0 063 264 85 264 89 0 038 0 057 31 268 02 268 20 0 035 0 054 268 81 268 83 0 052 0 064 268 74 268 78 0 045 0 059 32 271 91 272 10 0 031 0 056 272 69 272 72 0 042 0 053 272 63 272 68 0 040 0 058 33 275 87 276 03 0 035 0 054 276 65 276 69 0 045 0 068 276 58 276 63 0 046 0 054 34 279 72 279 90 0 038 0 055 280 54 280 58 0 049 0 072 280 47 280 51 0 049 0 062 35 283 52 283 71 0 044 0 062 284 33 284 37 0 037 0 063 284 26 284 30 0 049 0 057 36 287 35 287 55 0 043 0 054 288 17 288 22 0 049 0 066 288 12 288 16 0 042 0 057 37 291 25 291 44 0 034 0 055 292 10 292 11 0 044 0 075 292 01 292 06 0 042 0 062 38 295 03 295 22 0 043 0 065 295 89 295 91 0 047 0 067 295 79 295 85 0 037 0 062 39 299 06 299 26 0 045 0 055 299 93 299 96 0 064 0 068 299 85 299 90 0 046 0 063 40 302 75 302 95 0 039 0 054 303 61 303 65 0 048 0 073 303 52 303 60 0 047 0 065 41 306 64 306 86 0 035 0 066 307 56 307 60 0 038 0 087 307 48 307 53 0 050 0 062 42 310 49 310 70 0 047 0 060 311 44 311 47 0 062 0 080 311 35 311 41 0 046 0 066 43 314 40 314 62 0 041 0 073 315 40 315 43 0 046 0 096 315 30 315 37 0 045 0 070 44 318 47 318 69 0 039 0 062 319 50 319 52 0 048 0 089 319 40 319 47 0 047 0 071 DY
27. 5 ng each The chimpanzee and gorilla DNA samples produced partial profiles within the 100 330 base pair region The remaining species tested did not yield reproducible detectable products Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Sensitivity Sensitivity SWGDAM Guideline The ability to obtain reliable results from a range of DNA quantities to include the upper and 3 3 lower limits of the assay should be evaluated SWGDAM December 2012 Effect of DNA In a casework workflow the optimal amount of input male DNA added to the Yfiler quantity on results Plus Kit should be between 0 5 and 1 0 ng for 30 cycles of amplification Figure 22 on and importance of P28e 84 The DNA sample should be quantitated prior to amplification using a system such as the Quantifiler HP Human Plus DNA Quantification Kit Part no 4482911 or the Quantifiler Trio DNA Quantification Kit Part no 4482910 The final DNA concentration should be in the range of 0 05 0 10 ng uL so that 0 5 1 0 ng of male DNA will be added to the PCR reaction in a volume of 10 uL If the sample contains degraded DNA amplification of additional DNA may be beneficial quantitation If too much male DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in the following e Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off s
28. 82 0 029 0 050 217 76 217 77 0 032 0 038 9 222 32 222 41 0 023 0 032 222 87 222 90 0 023 0 064 222 86 222 87 0 030 0 040 10 227 38 227 47 0 027 0 030 227 91 227 94 0 016 0 068 227 90 227 93 0 036 0 054 11 232 43 232 52 0 032 0 037 232 96 232 98 0 031 0 065 232 95 232 98 0 032 0 046 12 237 48 237 58 0 021 0 038 238 03 238 04 0 035 0 047 238 00 238 03 0 032 0 049 13 242 64 242 71 0 024 0 033 243 14 243 17 0 027 0 052 243 14 243 16 0 025 0 037 14 247 82 247 87 0 022 0 031 248 30 248 32 0 040 0 050 248 28 248 31 0 033 0 046 15 252 87 252 90 0 022 0 037 253 31 253 33 0 027 0 056 253 31 253 34 0 024 0 041 16 257 80 257 85 0 014 0 029 258 24 258 26 0 021 0 041 258 24 258 27 0 034 0 039 DYS439 6 149 93 150 01 0 016 0 027 150 28 150 31 0 021 0 046 150 23 150 24 0 028 0 039 7 154 05 154 12 0 020 0 036 154 38 154 41 0 033 0 055 154 32 154 34 0 034 0 041 8 158 15 158 23 0 031 0 036 158 51 158 52 0 031 0 052 158 43 158 46 0 025 0 046 9 162 21 162 30 0 024 0 034 162 54 162 59 0 036 0 046 162 49 162 50 0 036 0 043 10 166 33 166 41 0 023 0 029 166 65 166 69 0 033 0 039 166 60 166 62 0 026 0 044 11 170 27 170 36 0 022 0 032 170 60 170 64 0 039 0 054 170 56 170 59 0 027 0 045 12 174 30 174 39 0 020
29. A Gill P Kayser M Mayr W R et al 2006 DNA Commission of the International Society of Forensic Genetics ISFG An update of the recommendations on the use of Y STRs in forensic analysis Forensic Sci Int 157 187 97 Yfiler Plus PCR Amplification Kit User Guide 119 Bibliography 120 Gusmao L Gonzalez Neira A Pestoni C Brion M Lareu M V Carracedo A 1999 Robustness of the Y STRs DYS19 DYS389 I and II DYS390 and DYS393 optimization of a PCR pentaplex Forensic Sci Int 106 163 72 Hall A and Ballantyne J 2003 The development of an 18 locus Y STR system for forensic casework Anal Bioanal Chem 376 1234 46 Holt C Stauffer C Wallin J Lazaruk L Nguyen T Budowle B and Walsh P 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Johnson C L Warren J H Giles R C Staub R W 2003 Validation and uses of a Y chromosome STR 10 plex for forensic and paternity laboratories J Forensic Sci 2003 48 6 1260 8 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lazaruk K Walsh P S Oaks F Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on
30. Amplification Kit User Guide Overview M Product overview eeen eenen eee eee eee 9 m Workflow overview for casework samples ooo 13 m Workflow overview for database samples ussen 14 E Instrument and software overview eneen eneen 15 m Materials and equipment W K KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK K 17 Product overview Purpose Product description Substrates The Yfiler Plus PCR Amplification Kit is a 6 dye short tandem repeat STR multiplex assay optimized to allow amplification from multiple male specific sample types such as male male male female mixtures and direct PCR amplification from the following types of single source samples e Blood and buccal samples on treated paper substrates without the need for sample purification e Blood samples collected on untreated paper substrates and treated with Prep n Go Buffer e Buccal samples collected on swab substrates and treated with Prep n Go Buffer Yfiler Plus PCR Amplification Kit amplifies 27 Y STR loci in a single PCR amplification reaction The Yfiler Plus Kit contains all the necessary reagents for the amplification of human male specific genomic DNA The reagents are designed for use with the following Life Technologies instruments Applied Biosystems 3500 3500xL Genetic Analyzer e Applied Biosystems 3130 3130x Genetic Analyzer GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block e G
31. I for varying amounts of time The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point 2 ng of degraded DNA or 1 ng undegraded DNA was amplified using the Yfiler Plus Kit As the DNA became increasingly degraded the loci became undetectable according to size The loci failed to robustly amplify in the order of decreasing size as the extent of degradation progressed Figure 24 Yfiler Plus PCR Amplification Kit User Guide 85 5 Experiments and Results Stability Figure 24 Amplification of A3121 DNA samples sonicated and incubated with increasing doses of DNase Panels 1 2 3 and 4 correspond to 0 4 5 and 6 units of DNase I Note that the Y axis scale is magnified for more degraded samples which generate lower peak heights 10 EDI en zo zo oo zooo eto za ano 1w an 120 zo zo ow etno yal dha la il jl T E a d T Mark Sample for Deleton EI ze o 0 units DNase E Mark Sanple for Det a 4 units DNase IT Mark Sample for Deleton eme a 5 units DNase emo em E Y ETO E Mark Semple for Deleton 6 units DNase 86 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Mixture studies Mixture studies SWGDAM Guideline 3 8 Male female mix
32. alleles display a lower level of stutter relative to the longer alleles within each locus Alleles at the low high extreme end of the range have minimal data points due to poor representation in the Life Technologies data set Some of these alleles were not represented at all Each allele within a locus displays percent stutter that is reproducible e Stutter filter sets in GeneMapper ID X Software calculated as the mean stutter for the locus plus three standard deviations are shown in Table 3 Peaks in the stutter position that are above the stutter filter percentage specified inthe software are not filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Mixture studies on page 87 The measurement of percent stutter for alleles that are off scale may be unusually high due to artificial truncation of the main allele peak Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra Peaks in the electropherogram Table 3 Marker specific stutter filter percentages for Yfiler Plus Kit loci Erna ac d Minus stuttert Plus stuttert porn ene ae hi DYS576 15 15 3 38 DYS3891 9 16 3 45 DYS635 13 38 3 3 DYS3891I 18 79 3 73 DYS627 15 18 2 62 2 71 DYS460 11 65 4 27 DYS458 15 31 2 52 DYS19 12 68 3 72 10 1 3 42 YGATAH4
33. essentially free of reproducible dye artifacts within the kit s read region for commonly used analytical thresholds Additional reproducible DNA dependent artifacts have been characterized and documented on Table 4 It is important to consider possible noise and artifacts when interpreting data from the Yfiler Plus Kit on the Applied Biosystems 3500 3500xL and 3130 3130x Genetic Analyzers Table 4 DNA dependent artifacts Artifact Color Size Comment FAM270 Blue 270 271 Minor cross reactive product observed with female DNA in excess of 2 yg FAM280 Blue 280 281 Minor cross reactive product observed with female DNA in excess of 2 ug FAM348 Blue 348 349 Specific to cell line derived kit Control DNA Y391 n 10 Green n 1Ont Specific to DYS391 Minor cross reactive product observed with male DNA in excess of 1 0 ng TAZ140 Red 139 140 Minor cross reactive product observed with female DNA in excess of 2 ug TAZ144 Red 144 145 Minor cross reactive product observed with female DNA in excess of 2 ug TAZ225 260 Red 225 260 Multiple minor cross reactive products observed with female DNA in excess of 2 ug TAZ412 Red 412 413 Cross reactive product observed with female DNA in excess of 100 ng Occurs outside of the read region Does not impact interpretation VIC70 Green 70 Sporadic PCR artifact Occurs outside of the VIC read region Does not impact interpretation Yfiler
34. for the markers DYS518 and DYS449 2014 Forensic Sci Int Genet doi 10 1016 j fsigen 2014 04 009 PubMed PMID 24854343 Nakahori Y Takenaka O and Nakagome Y 1991 A human X Y homologous region encodes amelogenin Genomics 9 264 269 Prinz M Ishii A Coleman A Baum H J Shaler R C 2001 Validation and casework application of a Y chromosome specific STR multiplex Forensic Sci Int 120 177 88 Yfiler Plus PCR Amplification Kit User Guide Bibliography Redd A J Agellon A B Kearney V A Contreras V A Karafet T Park H de Knijff P Butler J M Hammer M F 2002 Forensic value of 14 novel STRs on the human Y chromosome Forensic Sci Int 130 97 111 Schoske R Vallone P M Kline M C Redman J W Butler J M 2004 High throughput Y STR typing of U S populations with 27 regions of the Y chromosome using two multiplex PCR assays Forensic Sci Int 139 107 21 Scientific Working Group on DNA Analysis Methods GSWGDAM 2012 Validation Guidelines for DNA Analysis Methods Available at http swgdam org SWGDAM_Validation_Guidelines_APPROVED_Dec_2012 pdf Accessed 29 July 2013 Scientific Working Group on DNA Analysis Methods GWGDAM 2014 Interpretation Guidelines for Y Chromosome STR Typing by Forensic DNA Laboratories Available at http swgdam org SWGDAM_YSTR_Guidelines_APPROVED_01092014_v_02112014_FINAL pdf Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at t
35. has been observed in certain Y STR loci that include more complex nucleotide sequences including regions of dinucleotide repeats as shown in Figure 18 on page 77 In cases where these stutter peaks exceed the peak amplitude threshold e g 175 RFU they may be detected by analysis software as additional alleles in the profile GeneMapper ID X analysis files supplied for use with the Yfiler Plus Kit contain a minus 2 nt stutter filter for DYS19 DYS481 DYS533 and DYS627 to prevent these peaks from being called in normal profiles The proportion of the stutter product relative to the main allele stutter percent is measured by dividing the height of the stutter peak by the height of the main allele peak Such measurements have been made at Life Technologies for amplified samples at the loci used in the Yfiler Plus Kit All data were generated on the 3500xL Genetic Analyzer The stutter measurements were derived from DNA extracted from blood samples acquired from the Interstate Blood Bank Memphis Tennessee and Boca Biolistics Coconut Creek Florida The samples were collected in the United States with no geographical preference from randomly selected individuals of known ethnicities Some of the general conclusions from these measurements and observations are as follows For each Yfiler Plus Kit locus the stutter percent generally increases with allele length as shown in Figure 11 through Figure 17 on the following pages Smaller
36. ng in a maximum input volume of 10 uL for 30 PCR cycles If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in e Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data are problematic because Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate and it results in poor spectral separation pull up e Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile Methods of Life Technologies provides several kits for quantifying DNA in samples See the quantifying DNA references cited in the following table for details about these kits 20 Yfiler Plus PCR Amplification Kit User Guide Section 2 1 Amplification from extracted DNA DNA quantification Product Description Quantifiler Human DNA Quantification Kit Part no 4343895 and Quantifiler Y Human Male DNA Quantification Kit Part no 4343906 For more information see Quantifiler Human DNA Quantification Kits User s Manual Pub no 4344790 Prop
37. right side of main allele peaks and is therefore to be avoided Figure 19 Time course of 7 12 17 and 22 minutes during the final extension step A mixture of 1 ng of male DNA and 1 ug of female DNA was amplified with increasing final extension times resulting in complete A addition at the DYS438 locus and the disappearance of the OL A shoulder peak 230 240 250 4000 j 7 minutes 12 minutes 230 240 250 17 minutes biel 22 En 22 minutes 12 Lack of full A nucleotide addition may be observed in Yfiler Plus Kit results when the amount of input DNA is greater than recommended protocols This is because more time is needed for the DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA will also result in off scale data Yfiler Plus PCR Amplification Kit User Guide About artifacts Other DNA dependent artifacts Chapter 5 Experiments and Results Extra Peaks in the electropherogram Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible while anomalies are non reproducible intermittent occurrences that are not observed consistently in a system for example spikes and baseline noise Due to improvements in PCR primer manufacturing processes the incidence of artifacts has been greatly reduced in the Yfiler Plus PCR Amplification Kit Kit electropherograms are
38. sizing precision in a capillary electrophoresis instrument Electrophoresis 19 86 93 Li H Schmidt L Wei M H Hustad T Leman M I Zbar B and Tory K 1993 Three tetranucleotide polymorphisms for loci D3S1352 D351358 D351359 Hum Mol Genet 2 1327 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J I Lowe A L Ghosh S and Collins F S 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 709 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill P D Budowle B and McCord B R 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 Mills K A Even D and Murrau J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 Moretti T Baumstark A Defenbaugh D Keys K Smerick J and Budowle B 2001 Validation of short tandem repeats STRs for forensic usage Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples J Forensic Sci 46 3 647 660 Mulero J Ballantyne J Ballantyne K Budowle B Coble M Gusmao L Roewer L Kayser M Nomenclature update and allele repeat structure
39. 0 032 0 059 32 365 98 366 06 0 034 0 042 366 01 366 04 0 038 0 055 366 08 366 10 0 035 0 041 33 369 99 370 07 0 029 0 042 370 00 370 04 0 038 0 048 370 08 370 11 0 030 0 047 34 373 99 374 09 0 025 0 044 374 02 374 04 0 027 0 061 374 09 374 12 0 033 0 053 35 377 99 378 08 0 032 0 047 378 01 378 06 0 040 0 055 378 10 378 11 0 039 0 047 36 382 03 382 12 0 034 0 043 382 03 382 08 0 027 0 053 382 12 382 13 0 031 0 051 37 386 08 386 18 0 022 0 043 386 09 386 12 0 025 0 045 386 17 386 19 0 034 0 048 38 390 12 390 22 0 020 0 042 390 14 390 16 0 025 0 044 390 21 390 23 0 036 0 044 39 394 14 394 28 0 030 0 043 394 18 394 20 0 029 0 049 394 24 394 26 0 037 0 043 40 398 17 398 31 0 029 0 053 398 21 398 25 0 024 0 047 398 27 398 29 0 030 0 038 DYS456 10 76 25 76 30 0 033 0 043 76 25 76 28 0 044 0 050 76 08 76 10 0 031 0 042 11 80 51 80 56 0 016 0 036 80 54 80 56 0 025 0 047 80 40 80 42 0 026 0 037 12 84 72 84 78 0 023 0 035 84 74 84 77 0 009 0 043 84 63 84 64 0 028 0 035 13 88 92 88 97 0 020 0 036 88 95 88 99 0 021 0 035 88 84 88 85 0 024 0 036 14 93 11 93 16 0 022 0 036 93 13 93 17 0 024 0 047 93 03 93 05 0 028 0 039 98 Yfiler Plus PCR Amplification Kit User Guide Appendix ATable of Precision Results
40. 0 032 174 64 174 67 0 029 0 050 174 58 174 61 0 028 0 045 13 178 38 178 47 0 027 0 047 178 72 178 75 0 032 0 052 178 66 178 70 0 041 0 046 14 182 44 182 52 0 029 0 035 182 78 182 81 0 035 0 051 182 74 182 76 0 025 0 045 15 186 45 186 53 0 024 0 033 186 79 186 82 0 029 0 052 186 76 186 78 0 036 0 044 16 190 51 190 60 0 025 0 042 190 86 190 88 0 026 0 048 190 83 190 85 0 037 0 047 17 194 58 194 66 0 024 0 040 194 93 194 96 0 027 0 054 194 90 194 93 0 034 0 047 DYS448 14 277 79 277 88 0 032 0 045 278 38 278 41 0 033 0 059 278 39 278 41 0 040 0 063 Yfiler Plus PCR Amplification Kit User Guide 97 Table of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard Del deviation mean deviation mean deviation 15 283 72 283 80 0 021 0 044 284 33 284 34 0 030 0 065 284 31 284 34 0 034 0 048 16 289 61 289 69 0 033 0 043 290 22 290 25 0 031 0 062 290 21 290 24 0 031 0 050 17 295 50 295 59 0 031 0 045 296 12 296 14 0 039 0 060 296 11 296 13 0 039 0 059 18 301 34 301 43 0 036 0 043 301 98 302 00 0 040 0 061 301 95 302 00 0 040 0 066 19 307 17 307 28 0 035 0 043 307 85 307 88 0 042 0 061 307 83 307 87 0 037 0
41. 0 041 96 26 96 30 0 030 0 049 96 11 96 13 0 030 0 040 12 100 46 100 48 0 023 0 045 100 44 100 48 0 039 0 048 100 28 100 30 0 034 0 042 13 104 65 104 67 0 028 0 035 104 64 104 67 0 030 0 038 104 48 104 50 0 025 0 039 14 108 80 108 84 0 028 0 039 108 79 108 82 0 025 0 040 108 63 108 66 0 027 0 033 DYS481 17 206 82 206 84 0 018 0 034 206 89 206 93 0 023 0 040 206 96 206 97 0 025 0 038 Yfiler Plus PCR Amplification Kit User Guide 99 Table of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard Mean deviation mean deviation mean deviation 18 209 80 209 81 0 018 0 032 209 86 209 88 0 027 0 044 209 92 209 93 0 030 0 036 19 212 76 212 78 0 026 0 036 212 86 212 87 0 009 0 050 212 89 212 91 0 037 0 045 20 215 78 215 80 0 021 0 035 215 86 215 88 0 016 0 042 215 91 215 94 0 027 0 042 21 218 85 218 87 0 018 0 033 218 91 218 94 0 010 0 032 218 98 218 99 0 029 0 040 22 221 88 221 90 0 024 0 039 221 93 221 96 0 020 0 042 222 00 222 02 0 038 0 048 23 224 88 224 90 0 023 0 033 224 94 224 97 0 025 0 037 225 02 225 03 0 039 0 049 24 227 88 227 90 0 021 0 032 227 95 227 97 0 037 0 055 228 03 228 04 0 037 0 043 25
42. 0 045 Yfiler Plus PCR Amplification Kit User Guide 103 Table of Precision Results 104 Yfiler Plus PCR Amplification Kit User Guide Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis Table 9 Troubleshooting Observation Possible causes Recommended actions Incorrect volume or absence of Master Mix or Primer Set Faint or no signal from both the DNA Repeat amplification Control 007 and the DNA test samples at all loci No activation of DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 1 minute Master Mix not vortexed thoroughly before aliquoting Vortex the Master Mix thoroughly Primer Set exposed to too much light Store the Primer Set protected from light Evaporation Ensure that the plate is properly sealed with film and that you used a compression pad with the 9700 thermal cycler a compression pad is not needed with the Veriti thermal cycler PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Use of incorrect thermal cycling parameters MicroAmp Base used with tray retainer set and tubes in GeneAmp 9700 Check the protocol for correct thermal cycling parameters Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected Prepar
43. 0 726 0 459 0 632 0 703 YGATAH4 0 590 0 585 0 580 0 606 DYS448 0 707 0 583 0 697 0 755 DYS391 0 445 0 540 0 561 0 437 DYS456 0 615 0 737 0 700 0 603 DYS390 0 646 0 684 0 656 0 699 DYS438 0 551 0 581 0 688 0 547 DYS392 0 445 0 592 0 664 0 710 DYS518 0 843 0 806 0 807 0 867 DYS570 0 806 0 738 0 799 0 820 DYS437 0 504 0 577 0 592 0 476 DYS385 0 942 0 854 0 904 0 973 DYS449 0 857 0 783 0 818 0 882 DYS393 0 587 0 363 0 442 0 662 DYS439 0 629 0 625 0 682 0 669 DYS481 0 857 0 724 0 790 0 821 DYF387S1 0 941 0 874 0 913 0 945 DYS533 0 598 0 576 0 591 0 644 Gene diversity D 1 Ep where n sample size p allele frequency Johnson et al 2003 7 In addition to the alleles that were observed and recorded in the Life Technologies databases other known alleles have been published or reported to us by other laboratories Some of these alleles occur at a low frequency and include several microvariants Furedi et al 1999 Schoske et al 2004 Analyzing the population data 90 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Mutation rate Discriminatory capacity of haplotypes Table 7 shows the discriminatory capacity DC and the number of unique haplotypes UH for each Y STR marker combination listed The discriminatory capacity was determined by dividing the number of different haplotypes by the number of samples in that population Schoske et al 2004 A unique haplotype is defined as one tha
44. 036 0 040 13 84 65 84 77 0 021 0 033 85 27 85 30 0 040 0 052 84 98 85 01 0 031 0 037 14 88 71 88 86 0 029 0 041 89 35 89 38 0 040 0 068 89 07 89 10 0 029 0 040 15 92 79 92 93 0 026 0 043 93 42 93 47 0 039 0 057 93 17 93 19 0 024 0 040 16 96 84 96 99 0 029 0 039 97 49 97 54 0 041 0 055 97 24 97 26 0 029 0 040 Yfiler Plus PCR Amplification Kit User Guide 101 Table of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard mean deviation mean deviation mean deviation 17 100 90 101 05 0 021 0 045 101 59 101 63 0 027 0 051 101 34 101 35 0 038 0 039 18 104 98 105 14 0 029 0 040 105 69 105 72 0 038 0 069 105 41 105 44 0 026 0 037 19 109 02 109 19 0 026 0 053 109 73 109 77 0 033 0 071 109 47 109 49 0 025 0 043 20 113 02 113 20 0 025 0 050 113 76 113 79 0 037 0 080 113 49 113 50 0 034 0 040 21 116 93 117 09 0 029 0 042 117 65 117 67 0 033 0 065 117 38 117 40 0 031 0 040 22 120 77 120 95 0 025 0 042 121 51 121 54 0 042 0 074 121 23 121 27 0 026 0 041 23 124 66 124 83 0 029 0 042 125 43 125 46 0 041 0 081 125 14 125 17 0 030 0 039 24 128 56 128 74 0 030 0 050 129 36 129 38 0 062 0 069 129 07 129 09 0 028 0 040 25 132 47 1
45. 1 22 23 24 25 26 27 28 DYS570 DYS449 Red 403 0 30 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 DYS437 DYS481 DYS393 Purple 139 36 13 7 8 9 10 11 12 13 14 15 16 17 18 DYF38751 DYS533 w E E DYS385 DYS439 Purple 199 6 12 6 7 8 9 10 11 12 13 14 15 16 17 B B R S ble Spal Gl sul 5 E 8 CP J N J TJ DYS449 DY5481 Purple 256 0 22 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 DY5393 DYF38751 Purple nN 325 0 35 37 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 DYS439 DYS533 Purple bi 383 9 13 7 8 9 10 11 12 13 14 15 16 17 8 Import Yfiler Plus Stutter_v1 a b 50 Select the Yfiler_Plus_ Panels _v1 folder in the navigation panel Select File gt Import Marker Stutter to open the Import Marker Stutter dialog box Navigate to then open the Yfiler Plus Analysis files_v1X folder Select Yfiler Plus Stutter v1 then click Import Note Importing this file associates the marker stutter ratio with the bin set in the Yfiler_Plus_Panels_v1 folder and overwrites any existing stutter ratios associated with the panels and bins in that folder LJ Yfiler_Plus_Bins_v1 t L Yfiler_Plus_Panels_v1 te er Yfiler_Plus_Stutter_v1 txt All Files Yfiler Plus PCR Amplification Kit User
46. 10 225 12 0 022 0 050 225 07 225 09 0 036 0 048 7 229 25 229 33 0 027 0 041 229 12 229 14 0 028 0 044 229 08 229 11 0 042 0 047 8 233 37 233 44 0 026 0 042 233 21 233 24 0 026 0 045 233 19 233 21 0 038 0 048 9 237 40 237 47 0 028 0 037 237 24 237 26 0 023 0 066 237 22 237 25 0 033 0 046 10 241 50 241 57 0 026 0 049 241 31 241 34 0 035 0 052 241 30 241 31 0 023 0 038 11 245 53 245 61 0 024 0 040 245 37 245 38 0 022 0 046 245 34 245 37 0 031 0 046 12 249 71 249 81 0 027 0 038 249 53 249 55 0 005 0 034 249 51 249 54 0 034 0 043 13 253 74 253 84 0 025 0 037 253 55 253 57 0 021 0 040 253 55 253 57 0 030 0 046 14 257 69 257 79 0 027 0 047 257 50 257 52 0 031 0 047 257 51 257 52 0 028 0 041 15 261 66 261 75 0 023 0 039 261 46 261 49 0 031 0 040 261 48 261 50 0 033 0 043 16 265 67 265 76 0 027 0 041 265 50 265 51 0 018 0 044 265 50 265 52 0 030 0 046 17 269 72 269 82 0 022 0 047 269 54 269 58 0 022 0 042 269 55 269 57 0 036 0 046 18 273 73 273 83 0 026 0 045 273 54 273 58 0 027 0 055 273 54 273 57 0 029 0 047 19 277 83 277 93 0 025 0 046 277 64 277 66 0 040 0 058 277 65 277 67 0 031 0 042 20 281 85 281 95 0 025 0 044 281 66 281 70 0 035 0 044 281 67 281 69 0 033 0 044 21 285 85 285 94 0 029 0 044 285
47. 11 12 13 14 15 16 17 12 DYS481 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 22 DYF387S1 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 35 37 DYS533 7 8 9 10 11 12 13 14 15 16 17 13 10 Yfiler Plus PCR Amplification Kit User Guide Chapter 1 Overview 1 Product overview Allelic ladder Figure 1 shows the allelic ladder for the Yfiler Plus Kit See Allelic ladder profile requirements on page 33 for information on ensuring accurate genoty ping Figure 1 GeneMapper D X Software v1 4 plot of the Yfiler Plus Allelic Ladder zoo 140 180 220 260 ET Er E1 20 EEEE EAE N MIR M MR METIR Yfiler Plus PCR Amplification Kit User Guide 11 Overview Product overview Control DNA 007 profile 12 Figure 2 shows amplification of Control DNA 007 using the Yfiler Plus Kit Figure 2 1 ng of Control DNA 007 amplified with the Yfiler Plus Kit and analyzed on the Applied Biosystems 3500xL Genetic Analyzer E Mark Sample for Deletio HE DD DERDE DUET pe eee r a en E 260 EJ sa d 0 ll ll J o E E El E Merk Sample for Deletio mmm NAM SSS 1an den E Ed EJ 20 2000 ra B E Mark Sample for Deletio ma TT ZI dan 460 E En wo 12000 E E Mark Sample for Deletio mar O 1e 180 2 Ed E 200 Mark Sample for Deleto mea 2x2
48. 11 53 2 27 DYS448 4 68 2 29 DYS391 9 99 3 41 DYS456 15 36 3 74 DYS390 13 58 3 51 DYS438 5 86 2 76 DYS392 16 94 11 DYS518 25 5 4 85 DYS570 15 65 2 88 DYS437 8 13 1 65 DYS385 18 32 3 7 DYS449 23 24 4 2 DYS393 14 07 4 95 DYS439 9 89 3 39 DYS481 28 55 5 59 9 55 DYF387S1 15 71 NA DYS533 12 4 6 1 88 The stutter filters are displayed as percentages in the GeneMapper ID X Yfiler_Plus_Stutter txt file j Undetermined IMPORTANT The values in Table 3 were determined by Life Technologies during the developmental validation studies We recommend that laboratories perform their own internal validation studies to determine the appropriate values to use Yfiler Plus PCR Amplification Kit User Guide 71 72 Experiments and Results Extra Peaks in the electropherogram Figure 11 Minus stutter percentages for the DYF387S1 DYS19 and DYS385 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively Percent Stutter 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 emmae eee ccc o cements ew e e omme o oommammmsamsosn ooo o oo o so mus co o 29 30 3132 33 34 35 36 37 38 39 40 4142 43 44 45 DYF387S1 8 9 101112131415 16 17 18 19 20 5 6 7 8 9 10 1112 13 14 15 16
49. 20 DNA quantification sess sya kk sami kK ber iee tini Wide Rd de Sle 20 Prepare the amplification kit reactions oon 22 Perform PER woa gee ace ieee rene dader ad ed 23 m Section 22 Direct amplification of DNA snee 25 Optimize PCR cycle number oon 25 Treated or untreated paper prepare reactions 0 cece eee eee 26 Swab substrates prepare reactions KK eee KK KK KK eens 28 Perform POR nt are Shei man Pete ale aga tn eerd an te dee 31 Yfiler Plus PCR Amplification Kit User Guide 19 e AB reagents Section 2 1 Amplification from extracted DNA Required user supplied reagents In addition to the Yfiler Plus Kit reagents the use of low TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You can prepare the buffer as described in the procedure below or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together e 10 mL of 1M Tris HCI pH 8 0 e 0 2 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature DNA quantification Importance of Quantifying the amount of DNA in a sample before amplification allows you to quantification determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction The optimum amount of DNA for the Yfiler Plus Kit is 1 0
50. 227 31 227 38 0 024 0 036 227 88 227 92 0 034 0 051 227 76 227 77 0 034 0 043 25 231 45 231 52 0 023 0 036 232 03 232 06 0 034 0 064 231 91 231 93 0 037 0 050 26 235 35 235 42 0 021 0 030 235 93 235 95 0 039 0 053 235 81 235 84 0 032 0 042 27 239 49 239 55 0 032 0 036 240 07 240 09 0 000 0 058 239 95 239 97 0 043 0 046 28 243 60 243 65 0 026 0 038 244 17 244 19 0 023 0 035 244 05 244 09 0 031 0 048 29 247 70 247 75 0 022 0 032 248 24 248 27 0 017 0 039 248 14 248 17 0 038 0 046 30 251 73 251 77 0 021 0 037 252 25 252 29 0 020 0 052 252 14 252 18 0 032 0 045 YGATAH4 8 235 91 235 96 0 022 0 032 236 18 236 22 0 031 0 055 236 15 236 16 0 030 0 047 9 239 92 239 96 0 023 0 036 240 19 240 22 0 031 0 065 240 16 240 18 0 028 0 055 10 244 04 244 09 0 023 0 030 244 31 244 34 0 033 0 047 244 27 244 28 0 030 0 046 11 248 14 248 15 0 018 0 033 248 39 248 41 0 041 0 052 248 35 248 36 0 032 0 047 12 252 15 252 17 0 020 0 034 252 37 252 39 0 020 0 040 252 35 252 37 0 031 0 040 13 256 07 256 10 0 024 0 039 256 31 256 33 0 029 0 039 256 28 256 30 0 027 0 042 14 259 97 260 00 0 015 0 032 260 24 260 27 0 000 0 049 260 19 260 23 0 032 0 047 15 263 94 263 99 0 019 0 042 264 22 264 25 0 019 0 040 264 20 264 22 0 034
51. 230 90 230 92 0 017 0 034 230 96 230 99 0 035 0 054 231 03 231 04 0 030 0 049 26 233 91 233 93 0 022 0 033 233 97 233 98 0 027 0 049 234 04 234 06 0 032 0 042 27 236 91 236 93 0 018 0 034 236 98 237 00 0 023 0 053 237 06 237 07 0 033 0 037 28 239 94 239 95 0 023 0 033 239 99 240 01 0 000 0 048 240 07 240 08 0 026 0 048 29 243 03 243 05 0 023 0 031 243 08 243 10 0 026 0 046 243 15 243 16 0 030 0 034 30 246 11 246 13 0 023 0 031 246 14 246 16 0 025 0 052 246 21 246 23 0 029 0 040 31 249 17 249 21 0 023 0 032 249 18 249 21 0 009 0 038 249 27 249 28 0 030 0 038 32 252 15 252 20 0 025 0 033 252 19 252 20 0 020 0 045 252 27 252 28 0 029 0 039 DYS518 32 332 35 332 38 0 025 0 054 332 32 332 35 0 027 0 045 332 32 332 35 0 038 0 046 33 336 40 336 41 0 029 0 050 336 35 336 38 0 024 0 045 336 36 336 39 0 030 0 050 34 340 43 340 48 0 026 0 047 340 36 340 40 0 005 0 044 340 39 340 41 0 005 0 041 35 344 52 344 59 0 025 0 051 344 45 344 49 0 032 0 051 344 48 344 50 0 035 0 052 36 348 60 348 70 0 024 0 047 348 54 348 57 0 011 0 044 348 56 348 59 0 027 0 042 37 352 67 352 76 0 025 0 059 352 60 352 64 0 033 0 053 352 63 352 65 0 029 0 042 38 356 74 356 82 0 026 0 054 356 69 356 71 0 038 0 049 356 70 356 73 0 038 0
52. 32 66 0 025 0 042 133 28 133 32 0 035 0 072 132 99 133 04 0 029 0 036 DYS627 11 323 89 324 05 0 049 0 059 325 01 325 03 0 048 0 094 324 93 324 98 0 052 0 066 12 327 79 327 94 0 043 0 066 328 92 328 97 0 038 0 095 328 84 328 89 0 058 0 074 13 331 66 331 84 0 033 0 053 332 81 332 85 0 053 0 080 332 73 332 77 0 048 0 070 14 335 53 335 71 0 046 0 062 336 67 336 72 0 045 0 086 336 61 336 65 0 055 0 063 15 339 38 339 53 0 040 0 056 340 53 340 57 0 054 0 086 340 45 340 50 0 044 0 070 16 343 29 343 42 0 040 0 055 344 42 344 47 0 025 0 103 344 35 344 42 0 048 0 056 17 347 21 347 32 0 040 0 060 348 36 348 40 0 050 0 101 348 28 348 33 0 047 0 059 18 351 11 351 22 0 037 0 061 352 27 352 31 0 052 0 097 352 19 352 24 0 050 0 071 19 355 07 355 22 0 047 0 062 356 26 356 29 0 050 0 098 356 17 356 23 0 046 0 063 20 358 90 359 06 0 047 0 060 360 11 360 17 0 048 0 114 360 02 360 10 0 053 0 072 21 362 69 362 85 0 038 0 064 363 92 363 94 0 059 0 092 363 83 363 90 0 053 0 066 22 366 53 366 69 0 049 0 070 367 77 367 80 0 040 0 095 367 69 367 75 0 051 0 067 23 370 36 370 53 0 045 0 064 371 62 371 66 0 044 0 075 371 53 371 61 0 054 0 064 24 374 21 374 37 0 054 0 069 375 47 375 52 0 074 0 077 375 39 375 46 0 062 0 070 25 3
53. 4 96 315 09 0 029 0 040 315 75 315 77 0 019 0 060 315 61 315 65 0 042 0 051 19 318 11 318 23 0 022 0 035 318 88 318 90 0 028 0 056 318 75 318 78 0 035 0 047 20 321 21 321 35 0 022 0 039 321 97 322 00 0 049 0 063 321 84 321 88 0 027 0 058 DYS393 7 90 33 90 36 0 021 0 032 90 35 90 39 0 027 0 037 90 23 90 25 0 029 0 037 8 94 35 94 36 0 024 0 037 94 37 94 42 0 020 0 032 94 27 94 28 0 033 0 041 9 98 51 98 53 0 020 0 030 98 53 98 56 0 028 0 039 98 43 98 44 0 040 0 044 10 102 63 102 64 0 014 0 029 102 67 102 70 0 021 0 052 102 56 102 57 0 033 0 040 11 106 89 106 90 0 019 0 027 106 94 106 96 0 026 0 037 106 82 106 84 0 029 0 037 12 110 79 110 81 0 024 0 030 110 85 110 88 0 039 0 056 110 74 110 76 0 031 0 041 13 114 80 114 81 0 017 0 028 114 87 114 91 0 020 0 041 114 76 114 78 0 026 0 035 14 118 72 118 74 0 021 0 029 118 79 118 81 0 033 0 043 118 67 118 69 0 010 0 032 15 122 51 122 53 0 024 0 036 122 61 122 62 0 031 0 044 122 48 122 49 0 030 0 041 16 126 56 126 59 0 017 0 029 126 66 126 68 0 029 0 046 126 54 126 56 0 036 0 041 17 130 54 130 55 0 015 0 033 130 62 130 66 0 023 0 036 130 51 130 53 0 027 0 041 18 134 51 134 54 0 019 0 029 134 61 134 64 0 025 0 038 134 52 134 53 0 030 0 043 DYS437 96 Yfiler P
54. 4 samples for 3500 3500xL Genetic Analyzers 4393715 GeneScan 600 LIZ Size Standard v2 0 4408399 DS 36 Matrix Standard Kit Dye Set J6 4425042 Conditioning reagent 4393718 8 Capillary array 36 cm for 3500 Genetic Analyzers 4404683 24 Capillary array 36 cm for 3500xL Genetic Analyzers 4404687 96 well retainer amp base set Standard 3500 3500xL Genetic Analyzers 4410228 110 Yfiler Plus PCR Amplification Kit User Guide Appendix C Ordering information Equipment and materials not included Itemt Source 8 Tube retainer amp base set Standard for 3500 3500xL Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container 3500 series 4410715 For a complete list of parts and accessories for the 3500 3500xL instrument refer to the 3500 3500xL Genetic Analyzer User Guide Pub no 4401661 PCR amplification MicroAmp 96 Well Tray N8010541 MicroAmp Reaction Tube with Cap 0 2 mL N8010540 MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 8 Cap Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 MicroAmp Clear Adhesive Film 4306311 MicroAmp Optical Adhesive Film 4311971 MicroAmp Optical 96 Well Reaction Plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aer
55. 48 DYS449 DYS456 DYS458 DYS460 DYS481 DYS518 DYS533 DYS570 DYS576 DYS627 T T T T T T T DYS635 100 150 200 250 300 350 400 YGATAH4 Allele Size nt 1 0 ee 0 0 Size Deviation nt 0 5 x amp vy gt es x amp y zm e gt u e alo Sizing precision allows for determination of accurate and reliable genotypes Sizing precision was measured on the 3130x 3500 and 3500xL Genetic Analyzers The recommended method for genotyping is to use a 0 5 nt window around the size obtained for each allele in the Yfiler Plus Allelic Ladder A 0 5 nt window allows for the detection and correct assignment of alleles Any sample allele that sizes outside a window could be either of the following e An off ladder allele for example an allele of a size that is not represented in the Yfiler Plus Allelic Ladder e An allele that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument Table 8 on page 93 indicates typical precision results obtained from the sev
56. 5 The concentrations of humic acid tested were 0 100 and 250 ng uL The same concentrations were tested with the Yfiler Kit for comparison At 250 ng uL neither kit yielded amplified products Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 Stability Figure 23 Electropherograms for the Yfiler Plus and AmpF STR Yfiler Kits show the improved performance of the Yfiler Plus Kit in the presence of humic acid compared to the Yfiler Kit The Y axis scale is 0 to 20 000 RFUs for the top two panels 0 to 30 000 RFUs for the third panel and 0 to 4000 RFUs for the bottom panel T Marie Samie for Deletion Yfiler Plus Kit uninhibited control ak Lalik Add ov owe OPG AA aA iMh LAUA i TF Mark Sange for Delton Yfiler Plus Kit with 100 ng L humic acid dadh Uid ak ill Yfiler Kit uninhibited control Il l l l l mar Sane for Deleter Yfiler Kit with 100 ng yL humic acid Deg raded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This is due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for preferential amplification of loci High molecular weight DNA was incubated with the enzyme DNase
57. 6 17 1819 DYS393 9 10 1112 13 14 15 16 17 18 19 DYS437 5 6 7 8 9 101112131415 1617 DYS438 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra Peaks in the electropherogram Figure 14 Minus stutter percentages for the DYS439 DYS448 DYS449 and DYS456 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively Percent Stutter 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 5 6 7 8 9 101112131415161718 1314151617181920 2122232425 21222324 252627 282930 31323334 35 36 37 38 39 40 41 DYS439 DYS448 DYS449 9 1011121314 151617 181920 21222324 DYS456 Figure 15 Minus stutter percentages for the DYS458 DYS460 DYS481 and DYS518 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively Percent Stutter 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 111213 14 15 16 17 181920 21222324 25 DYS458 Yfiler Plus PCR Amplification Kit User Guide 6 7 8 9101112131415 DYS460 1617 18 1920 2122 2324 25 2627 282930 313233 DYS481 T 323334353637 383940 41424344454647 484950 DYS518 75 76 Experiments and Results Extra Peaks in the electropher
58. 65 285 69 0 036 0 043 285 67 285 68 0 030 0 043 22 289 82 289 93 0 029 0 052 289 63 289 65 0 037 0 050 289 63 289 66 0 030 0 040 23 293 78 293 89 0 031 0 046 293 58 293 62 0 030 0 040 293 58 293 60 0 032 0 047 24 297 79 297 89 0 029 0 042 297 57 297 60 0 037 0 065 297 56 297 60 0 034 0 050 25 301 75 301 85 0 025 0 052 301 56 301 58 0 038 0 045 301 55 301 57 0 020 0 050 26 305 71 305 82 0 034 0 039 305 51 305 53 0 018 0 054 305 51 305 53 0 026 0 039 27 309 71 309 82 0 036 0 044 309 50 309 55 0 028 0 042 309 52 309 54 0 032 0 044 28 313 78 313 87 0 032 0 051 313 53 313 58 0 005 0 051 313 55 313 58 0 043 0 049 DYS3891 9 146 74 146 78 0 025 0 034 146 98 147 01 0 019 0 044 146 81 146 84 0 025 0 042 10 150 83 150 87 0 026 0 033 151 06 151 09 0 019 0 039 150 90 150 91 0 032 0 041 11 154 86 154 91 0 024 0 031 155 09 155 11 0 030 0 042 154 92 154 94 0 034 0 038 12 158 98 159 04 0 027 0 036 159 22 159 24 0 007 0 030 159 04 159 07 0 021 0 035 13 163 19 163 26 0 020 0 031 163 41 163 43 0 028 0 045 163 25 163 27 0 026 0 038 14 167 12 167 19 0 018 0 034 167 33 167 35 0 015 0 030 167 18 167 19 0 027 0 033 15 171 17 171 21 0 017 0 040 171 34 171 37 0 022 0 039 171 21 171 22 0 021 0 038 16 175 19 175 24 0 020 0
59. 78 05 378 21 0 059 0 074 379 32 379 36 0 055 0 067 379 23 379 32 0 058 0 067 26 381 90 382 06 0 062 0 080 383 22 383 27 0 059 0 086 383 12 383 20 0 056 0 074 27 385 79 385 94 0 064 0 079 387 10 387 14 0 069 0 094 387 02 387 10 0 046 0 073 DYS635 15 191 34 191 39 0 026 0 034 191 90 191 92 0 036 0 052 191 76 191 77 0 034 0 046 16 195 40 195 44 0 024 0 038 195 95 195 98 0 030 0 049 195 82 195 83 0 033 0 047 17 199 45 199 49 0 025 0 036 200 00 200 01 0 000 0 056 199 87 199 88 0 042 0 046 18 203 42 203 46 0 020 0 036 203 96 203 98 0 021 0 048 203 82 203 83 0 031 0 044 19 207 37 207 41 0 021 0 038 207 92 207 94 0 022 0 040 207 77 207 79 0 030 0 040 20 211 35 211 38 0 027 0 036 211 89 211 91 0 017 0 060 211 75 211 77 0 034 0 041 21 215 41 215 44 0 022 0 034 215 96 215 99 0 016 0 059 215 82 215 83 0 030 0 052 22 219 43 219 48 0 027 0 039 219 99 220 01 0 000 0 048 219 85 219 87 0 045 0 046 102 Yfiler Plus PCR Amplification Kit User Guide Appendix ATable of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard mean deviation Mean deviation mean deviation 23 223 44 223 49 0 023 0 037 224 00 224 02 0 033 0 061 223 86 223 89 0 038 0 053 24
60. 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block 4314878 Silver 96 Well Sample Block N8050251 Gold plated Silver 96 Well Sample Block 4314443 Tabletop centrifuge with 96 Well Plate Adapters optional MLS major laboratory supplier Harris Micro Punch tool 1 2 mm MLS Copan NUCLEIC CARD collection device 4473980 Copan NUCLEIC CARD collection device 1 Spot 4474001 Copan NUCLEIC CARD Color 1 spot 4473974 CPA200 Semi Automated Punch Instrument with a 1 2 mm punch head CPA300 Fully Automated Punch Instrument with a 1 2 mm punch head Contact your local Life Technologies support representative for information 96 well deep well plate 4392904 Table 11 Software Software Source 3500 3500xL Data Collection Software v2 RUO 4475183 HID Updater 3500 Data Collection Software v2 44806701 3130 Data Collection Software v4 44751051 3130x Data Collection Software v4 4475126t 3130 3730 Data Collection Software v4 6 Dye Module v1 t GeneMapper D X Software v1 4 Full Installation 4479707 GeneMapper D X Software v1 4 Client Installation 4479711 t Contact your Life Technologies HID representative Yfiler Plus PCR Amplification Kit User Guide 109 Ordering information Equipment and materials not included Table 12 Other items Item So
61. Guide 4 Panel Manager Chapter 4 Analyze Data 4 Set up GeneMapper ID X Software for data analysis 9 View the imported marker stutters in the navigation pane a Double click the Yfiler_Plus_Panels_v1 folder to display its list of kits in the right pane b Double click the Yfiler_Plus_Panels_v1 folder to display its list of markers below it c Double click DYS576 then click Stutter Ratio amp Distance to display the Stutter Ratio amp Distance view for the marker in the right pane File Edit Bins View Help i Bl BE BE Bin Set vier Pus_pins_vi J l u u u e B Yfiler_Plus_Panel_v1 E DYS635 DYS389II G DYS627 DYS460 DYS458 DYS19 GATA_H4 DYS448 DYS391 DYS456 DYS390 DYS438 DYS392 DYS518 p e En r Ens En B Ens Ens Please enter the stutter filter s for DYS5 76 marker here If left blank the global stutter filter will be applied Minus Stutter Plus Stutter From Distance To Distance From Distance To Distance prs jes 4 75 1 3 25 4 75 2 3 4 New edt Delete Lox cancel _ Appiy Heb 10 Click Apply then OK to add the Yfiler Plus Kit panel bin set and marker stutter to the GeneMapper ID X Software database IMPORTANT If you close the Panel Manager without clicking Apply the panels bin sets and marker stutter w
62. K kk kK kk kk kK kK kK kk kk kk kk kk kk kk kk kk kk kk kk kK kk kk kk 113 Amplified DNA work area nnee KK kk kk kk kk kk kK kk kk kk kk kk kK kk kk kk 114 APPENDIX E Safety kk kk kk kK KK KK KK KK KK KK KK KK KK KK 115 Che micalisafety nn naer ener veer or ern IE a agree ese REE xua ve ebe sa A REN 116 Biological hazard safety rattan etna a kk A ee eed kk waa all lele EE RE wal kok A un dl kl RR 116 Bibliography Zeste Weerd ded Saye Qi eva ayar aya e ast 119 Documentation and Support J S kK KK EK KK RR RR KK KK kk kk a 123 Related documentation sny Sir kla Ana nad IA EER Y RE eenen 123 OBTAIN SDS EEEN e Ee oe kl n tema O a Dl a tee KO E ONA e K ke 123 Obtain Supporter vet setter noe REE Re 124 Limited Product Warranty eeen 124 INDER antennes neemen Sockets Gee des Aten De al an eee 125 Yfiler Plus PCR Amplification Kit User Guide About This Guide IMPORTANT Before using this product read and understand the information in the Safety appendix in this document Revision history Revision Date Description A July 2014 New document B October 2014 Add Chapter 5 Purpose The Yfiler Plus PCR Amplification Kit User Guide provides information about the Life Technologies instruments chemistries and software associated with the Yfiler Plus PCR Amplification Kit Yfiler Plus PCR Amplification Kit User Guide About This Guide Purpose 8 Yfiler Plus PCR
63. MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube Prepare the DNA samples Yfiler Plus PCR Amplification Kit User Guide Perform PCR Section 2 1 Amplification from extracted DNA 2 Perform PCR DNA sample To prepare Negative control Add 10 uL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 0 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 1 0 ng of total DNA is in a final volume of 10 uL Add 10 uL of the diluted sample to the reaction mix Positive control Add 007 control DNA to a total amount of 1 0 ng The final reaction volume sample or control plus reaction mixture is 25 uL Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes Centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders if using 96 well plates Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold plated silver 96 well block or a Veriti 96 Well Thermal Cycler Note The Yfiler Plus Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96 well block Use of this thermal cycling platform may adversely affect performance of the Yfiler Plus Kit Program the thermal cycling conditions When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode When usi
64. NA Typing Burlington MA Elsevier Academic Press Butler J M Schoske R Vallone P M Kline M C Redd A J Hammer M F 2002 A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers Forensic Sci Int 129 10 24 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 Edwards A Hammond H A Lin J Caskey C T and Chakraborty R 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Frank W Llewellyn B Fish P et al 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 642 646 Furedi S Woller J Padar Z Angyal M 1999 Y STR haploty ping in two Hungarian populations Int J Legal Med 113 38 42 Gonzalez Neira A Elmoznino M Lareu M V Sanchez Diz P Gusmao L Prinz M Carracedo A 2001 Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies Forensic Sci Int 122 19 26 Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S Iovannisci D M Woo S Winn Deen E S 1994 High density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Gusmao L Butler J M Carracedo
65. S19 9 183 90 183 94 0 023 0 030 184 04 184 06 0 028 0 048 183 99 184 01 0 026 0 039 10 188 07 188 11 0 022 0 036 188 20 188 22 0 026 0 035 188 16 188 18 0 026 0 040 11 192 11 192 13 0 026 0 036 192 26 192 28 0 026 0 040 192 22 192 24 0 025 0 037 12 196 10 196 13 0 018 0 039 196 24 196 27 0 032 0 046 196 21 196 23 0 032 0 041 13 200 17 200 21 0 019 0 034 200 30 200 33 0 029 0 046 200 28 200 29 0 037 0 043 14 204 09 204 13 0 020 0 036 204 24 204 27 0 027 0 038 204 21 204 21 0 029 0 037 15 208 05 208 09 0 018 0 028 208 22 208 24 0 029 0 043 208 16 208 18 0 027 0 038 16 212 03 212 05 0 023 0 032 212 20 212 26 0 015 0 053 212 15 212 16 0 024 0 046 17 216 07 216 10 0 023 0 032 216 22 216 25 0 016 0 052 216 18 216 20 0 029 0 044 Yfiler Plus PCR Amplification Kit User Guide 93 Table of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard zak deviation mean deviation mean deviation 18 220 14 220 19 0 020 0 034 220 30 220 34 0 033 0 046 220 27 220 29 0 020 0 043 19 224 15 224 20 0 024 0 035 224 34 224 35 0 040 0 058 224 30 224 31 0 036 0 047 DYS385 6 225 25 225 31 0 029 0 041 225
66. SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of 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 After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the 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 A WARNING Potential Biohazard Depending on the samples used on this instrument the surface m
67. USER GUIDE applied biosystems life technologies Yfiler Plus PCR Amplification Kit for use with 100 reaction kit Part no 4484678 500 reaction kit Part no 4482730 Publication Number 4485610 Revision B 9 technologies For Forensic or Paternity Use Only For Forensic or Paternity Use Only Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S 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 Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS 2014 Thermo Fisher Scientific Inc All rights reserved All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Windows and Windows Vista are registered trademarks of Microsoft Corporation
68. YS570 19 17 DYS437 14 15 DYS385 16 19 11 14 DYS449 29 30 DYS393 14 13 DYS439 11 12 DYS481 27 22 DYF387S1 36 39 35 37 DYS533 11 13 A representative electropherogram of 1 ng total male male DNA mixture studies is shown in Figure 26 The limit of detection is when the minor component is present at approximately one tenth of the concentration of the major component The limit of detection for the minor component is influenced by the combination of genotypes in the mixture 88 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Population data Figure 26 Mixtures of two male DNA samples 1 8 ratio 125 pg 875 pg 1 ng input DNA The alleles attributable to the minor component even when the major component shares an allele are highlighted E Mark Sample for Deletion DSR cc Ss m M j m m r bi Mark Sample for Deletion ose HEZ BIM TJ A Tir Mark Sample for Deletion Mark Sample for Deletion sa ro Ta ed Mark Sample for Deletion Jl In da Population data SWGDAM YSTR Guideline 10 1 Overview Population samples used in these studies Gene diversity values The laboratory should establish guidelines for the number of Y STR loci used for searches of population databases SWGDAM January 2014 All Y STR loci analyzed in commercial kits are physically linked o
69. a half swab Add 400 uL Prep n Go Buffer Part no 4471406 to 1 5 mL tubes or the appropriate wells of a 96 well deep well plate Part no 4392904 Into each tube or well put the entire head of each swab and let stand for 20 minutes at room temperature 20 to 25 C to lyse the sample After 20 minutes transfer the sample lysate out of the sample plate into tubes or plates for storage then discard the deep well plate containing the swab heads Note To minimize the risk of contamination do not remove the swab heads from the sample lysate plate before transferring the lysate Proceed to Prepare the reactions on page 29 or see Store the sample lysate on page 31 Prepare the This protocol may improve the performance for challenging or aged samples sample lysate heat protocol 2 28 Preheat the heat block to 90 C or the oven with metal plate adaptor to 99 C Add 400 uL Prep n Go Buffer for buccal swabs Part no 4471406 to 1 5 mL tubes or the appropriate wells of a 96 well deep well plate Part no 43929040 Yfiler Plus PCR Amplification Kit User Guide Section 2 2 Direct amplification of DNA Swab substrates prepare reactions 3 Into each tube or well put the entire head of each swab If you are using tubes cap the tubes Let the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample 4 After 20 minutes remove the tubes or the deep well plate f
70. an deviation Mean deviation mean deviation 8 342 44 342 48 0 024 0 040 342 64 342 69 0 027 0 043 342 61 342 62 0 040 0 046 9 346 54 346 56 0 026 0 038 346 72 346 77 0 028 0 044 346 68 346 70 0 032 0 047 10 350 61 350 63 0 026 0 039 350 82 350 85 0 026 0 053 350 75 350 76 0 033 0 048 11 354 68 354 69 0 024 0 035 354 87 354 91 0 024 0 037 354 81 354 83 0 028 0 051 12 358 73 358 77 0 026 0 035 358 93 358 97 0 015 0 059 358 88 358 90 0 029 0 046 13 362 76 362 78 0 023 0 035 362 96 362 99 0 018 0 043 362 90 362 93 0 028 0 045 14 366 76 366 79 0 021 0 035 366 95 366 99 0 029 0 048 366 92 366 95 0 034 0 043 15 370 78 370 82 0 022 0 029 370 99 371 01 0 032 0 064 370 94 370 97 0 031 0 048 16 374 79 374 82 0 033 0 034 374 99 375 02 0 030 0 047 374 93 374 96 0 035 0 047 17 378 80 378 83 0 025 0 039 379 01 379 03 0 010 0 045 378 94 378 97 0 041 0 049 DYS570 10 97 99 98 02 0 021 0 034 98 02 98 04 0 026 0 044 97 95 97 96 0 035 0 043 11 102 12 102 15 0 024 0 034 102 13 102 16 0 023 0 041 102 08 102 09 0 028 0 034 12 106 23 106 27 0 021 0 029 106 26 106 29 0 032 0 046 106 20 106 21 0 026 0 036 13 110 33 110 35 0 022 0 033 110 35 110 37 0 032 0 044 110 28 110 30 0 028 0 043 14 114 36 114 38 0 018 0 030 114 40 114 41 0 024
71. are The instructions and examples in this section refer to the latest version of panel bin and stutter file available at the time of publication Before using the Before you use GeneMapper ID X Software v1 4 to analyze data for the first time you software for the must do the following first time 1 Check www lifetechnologies com support gt Software Patches amp Check panel bin 1 and stutter file version Updates GeneMapper ID X Software to obtain the latest Yfiler Plus Kit panel bin and stutter files Download and import the files into the GeneMapper ID X Software as explained in Import panels bins and marker stutter on page 48 Note When downloading new versions of analysis files refer to the associated Read Me file for details of changes between software file versions If you have validated previous file versions for data analysis conduct the appropriate internal verification studies before using new file versions for operational analysis Create an analysis method as explained in Create an analysis method on page 52 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information Define custom views of plots Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information Start the GeneMapper ID X Software then lo
72. ay adversely affect performance of the Yfiler Plus Kit e Veriti 96 Well Thermal Cycler 114 Yfiler Plus PCR Amplification Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Yfiler Plus PCR Amplification Kit User Guide 115 E Safety Chemical safety Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain
73. ay be considered a biohazard Use appropriate decontamination methods when working with biohazards 116 Yfiler Plus PCR Amplification Kit User Guide Appendix E Safety E Biological hazard safety AN WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals 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 In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional infor
74. ays The primary quantification targets Small Autosomal and Y consist of relatively short amplicons 75 to 80 bases to improve the detection of degraded DNA samples In addition the kit each contains a Large Autosomal target with a longer amplicon gt 200 bases to aid in determining if a DNA sample is degraded Yfiler Plus PCR Amplification Kit User Guide 21 2 Perform PCR Prepare the amplification kit reactions Product Description Quantifiler Trio DNA Quantification Kit Cat no 4482910 For more information see Quantifiler HP and Trio DNA Quantification Kits User Guide Pub no 4485354 Properties The Quantifiler Trio Kit is designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample How it works The Quantifiler Trio DNA Quantification Kit uses multiple copy target loci for improved detection sensitivity The human specific target loci Small Autosomal Large Autosomal and Y chromosome targets each consist of multiple copies dispersed on various autosomal chromosomes Small Autosomal and Large Autosomal or multiple copies on the Y chromosome To maximize the consistency of quantification results genomic targets were selected with conserved primer and probe binding sites within individual genomes and also with minimal copy number variability between different individuals and population groups As a result the detection sensitivity of the assa
75. c 3500xL8 Collection 3500 DC v2 0 listed above Analyzer User Guide Software v2 0 Pub no 4476988 ee el Ae HID Updater 3500 Data Collection Software v2 Release Notes t This kit was developed using an injection time of 16 seconds on the 3500 instrument This is different than the default injection time of 15 seconds The instrument protocol will need to be modified accordingly This kit was developed using two injection voltage conditions for the 3500 instrument 1 2 kV 16 sec and 1 5 kV 16 sec You are encouraged to explore both options during validation to determine which protocol provides the best results on your instrumentation 8 We conducted validation studies for the Yfiler Plus Kit using the 3130xl 3500 or 3500xL configurations tt This kit was developed using two injection voltage conditions for the 3500xL instrument 1 2 kV 24 sec and 1 5 kV 24 sec You are encouraged to explore both options during validation to determine which protocol provides the best results on your instrumentation Yfiler Plus PCR Amplification Kit User Guide 35 3 Perform electrophoresis Set up the 3500 3500xL instruments for electrophoresis Obtain and run the HID Updater Create a Yfiler Plus assay Modify 3500 QC protocol size calling method 36 You can run 6 dye samples on 3500 Data Collection Software v1 or v2 Before running on either system for the first time run the HID Updater 3500 DC v2 0 Part no 4480670 The HID Upda
76. cale data Off scale data is a problem for two reasons Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate which results in poor spectral separation pull up e Incomplete A nucleotide addition The sample can be re amplified using less DNA Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA This kit was developed using two injection conditions 3130xl 3 kV 10 sec and 3 kV 13 sec 3500 1 2 kV 16 sec and 1 5 kV 16 sec 3500xL 1 2 kV 24 sec and 1 5 kV 24 sec You are encouraged to explore both options during validation to determine which protocol provides the best results on your instrumentation The enhanced injection conditions resulted on average improvements in peak height of 25 Figure 22 on page 84 Note Please refer to Section 2 2 on page 25 to optimize the PCR cycle number used for direct amplification from multiple sample types and substrate combinations Yfiler Plus PCR Amplification Kit User Guide 83 5 Experiments and Results Stability Stability SWGDAM Guideline 3 4 Lack of amplification of some loci Effect of i
77. e Guide 4426482 GeneMapper ID X Software Version 1 4 User Bulletin 4477684 Portable document format PDF versions of this guide and the documents listed above are available at www lifetechnologies com Note To open the user documentation available from the our web site use the Adobe Acrobat Reader software available from www adobe com Obtain SDSs Safety Data Sheets SDSs are available from www lifetechnologies com sds Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Yfiler Plus PCR Amplification Kit User Guide 123 Documentation and Support Obtain support Obtain support For HID support In North America Send an email to HIDTechSupport lifetech 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 lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training e Download software updates and patches Limited Product Warrant
78. e PCR product as described in Prepare samples for electrophoresis on the 3500 3500xL instruments on page 38 or Prepare samples for electrophoresis on the 3130 3130xl instruments on page 43 Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide Yfiler Plus PCR Amplification Kit User Guide 105 El Troubleshooting Observation Possible causes Recommended actions Positive signal from Control DNA 007 but partial or no signal from DNA test samples Quantity of DNA test sample is below assay sensitivity Quantify DNA and when possible add 1 0 ng of DNA For low concentration samples add up to 10 uL of the DNA sample to the reaction mix see Prepare the amplification kit reactions on page 22 Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantify DNA and add minimum necessary volume Repeat test Wash the sample in Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degraded The Quantifiler HP and Trio Kits can help evaluate sample integrity during the quantification step If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Dilution of test DNA in water or wrong buffer for example TE formula with incorrect EDTA concentration Redilute DNA using low TE buffer
79. e version kk kk kk kK KK KK KK KK KK KK eee eee 47 Import panels bins and marker stutter kk kk kk kk kk kk kK KK cece ee KK eee KK KK ees 48 Create an analysis method eee eeen 52 General tab settings ooren 53 Allele tab settings Att heen ane nn et hbo eats 54 Peak Detector tab settings nennen 54 Yfiler Plus PCR Amplification Kit User Guide Contents Peak Quality tab settings 3 nnee eneen eenen 56 SQ ScGOstab settings L un lu tt ee hie Se ee Ka Ee Ki k ee a en le ke 57 Create a size standard kk kk kk kK kk kK kK kK KK KK KK KK KK kk kk kk eee 57 Analyze and edit sample files with GeneMapper D X Software enn 59 Examine andedit project iy s4 2 XW etn A Norte kalik ae Gere td lee ee 60 For more informations nrd en HRH H HH HHHH HHHH REAR AA LES 61 Em CHAPTER5 Experiments and Results WALA 0 cece eens 63 OVERVIEW redenen ereen ardea ed Rd Wee aaa 63 Importance of validation oenen 63 EXxperimentscoOmaitionss 2 lt 5 asten wett ant en ra a a ti A EN ER be 63 Developmental validation enen nennen 64 SWGDAM guideline 2 2 1 kk kk kk kk kk kK kk kk tent kk kk kk ee kk kk kk 64 SWGDAM quideline 3 9 2 zate hug decline begga e gaaat ek eee he dn Re a da aed 64 PGRICOMPONCMIS koca ats ke hay En aa 64 Thermal cycler parameters n dennen deepsea eg kk kk kk eae kk k 65 Accuracy precision and reproducibility kk kk kk kk kk kk kk KK kK KK KK KK KK KK KK KK
80. ecies e g detection of microbial DNA in a human assay should be determined SWGDAM December 2012 The Yfiler Plus Kit provides the required degree of specificity such that it is specific to primates Other species do not amplify for the loci tested Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The Yfiler Plus Kit provides the required degree of specificity for the species tested Figure 21 on page 82 Figure 21 Representative electropherograms from a species specificity study including positive and negative control JT Mark Semple for Deletion Male Sha Lu tud control TT Mark Sample for Deletion ri Chimp ilk j d i n Jet it a E Mark sample for Deletion Dog JT Mark Semple for Deletion Horse Mark Sample for Del wi Microbial ool S m e Negative ppntrol The following experiments were conducted to investigate interpretation of Yfiler Plus Kit results from nonhuman DNA sources The extracted DNA samples were amplified in Yfiler Plus Kit reactions and analyzed using the 3100 Genetic Analyzer e Primates Gorilla chimpanzee and macaque 1 0 ng each e Non primates Mouse dog pig rat sheep horse chicken and cow 10 ng each e Microorganisms Candida albicans Neisseria gonorrhoeae Escherichia coli 0157 H7 Bacillus subtilis Staphylococcus aureus and Lactobacillus rhamnosus
81. ee ees aa 2000 sen ooo HA Yfiler Plus PCR Amplification Kit User Guide Chapter 1 Overview 1 Workflow overview for casework samples Workflow overview for casework samples Prepare T Ss Be a samples E S em os a e m AutoMate Express System PrepFiler Express Kit SS ar EZ gt 2 N 2 ee j j Quantifiler Trio DNA Quantification Kit Perform PCR ve gg Yfiler Plus PCR Amplification Kit vs ag 5 ey QZ E 2 a GeneAmp PCR System 9700 Cycler Veriti 96 Well Thermal Cycler Perform m electro ol phoresis 3130 3130xl 3500 3500xL Genetic Analyzer Genetic Analyzer Analyze By data z GeneMapper D X v1 4 Yfiler Plus PCR Amplification Kit User Guide 13 1 Overview Workflow overview for database samples Workflow overview for database samples Prepare co c 2 samples Ta Treated or untreated paper substrates Ta Swab substrates 8E 8E n u 23 vg Sa os E a ag a n Lyse in kula CPA200 or CPA300 Prep n Go Buffer unc instrument v u Perform 55 BE PCR 25 Yfiler Plus PCR Amplification Kit 0 ag GeneAmp PCR System 9700 Cycler Veriti 96 Well Thermal Cycler Perform PCR Perf Perform ode 3 phoresis 3130 3130xl 3500 3500xL Genetic Analyzer Genetic Analyzer Analyze data ev GeneMapper ID X v1 4 14 Yfiler Plus PCR Amplification Kit User Guide Instrument and software overview
82. ee ie et treaties tn eet Gell te dn he eee 84 Mixtuire studiess ii ee tenten eb AAR Pee ha tiie 87 Population datas nen sanne k ne Waite yo eae Semen pe Orel kn k le eae 89 Mutation tate cee ee b ee ee Ede Added 91 This chapter provides results of the developmental validation experiments we performed using the Yfiler Plus PCR Amplification Kit Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 Wallin et al 1998 We performed experiments to evaluate the performance of the Yfiler Plus Kit according to the updated and revised guidelines from the Scientific Working Group on DNA Analysis Methods SWGDAM December 2012 Based on these guidelines we conducted experiments that comply with guidelines 2 0 and 3 0 and its associated subsections This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2000 This chapter will discuss many of the experiments we performed and examples of the results we obtained We chose conditions that produced optimum PCR product yield
83. elect the sample type Analysis Method Yfiler_Plus_AnalysisMethod_v1 or the name of the analysis method you created Panel Yfiler_Plus_Panels_v1 or the version installed Size Standard Use a size range of 60 460 bp for Local Southern size calling method t t The Yfiler Plus Kit was originally validated using the GeneScan 600 LIZ Size Standard v2 0 If you use a different size standard perform the appropriate internal validation studies to support the use of this size standard with the Yfiler Plus Kit Note For more information about how the Size Caller works refer to the GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 Yfiler Plus PCR Amplification Kit User Guide 59 Analyze Data Examine and edit a project 3 Click Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis as a completion bar extending to the right with the percentage indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Analysis Summary tab is displayed and the Genotypes tab becomes available upon completion of the analysis Analysis summary window after analysis File Edit Analysis View Tools Admin Help GOE Ww EI BB am
84. en injections of the Yfiler Plus Allelic Ladder analyzed on the 3130x 3500 and 3500xL Genetic Analyzers 36 cm capillary and POP 4 polymer The size standard used was GeneScan 600 LIZ Size Standard v2 0 The results were obtained within a set of injections on a single capillary array As indicated above sample alleles may occasionally size outside of the 0 5 nt window for a respective allelic ladder allele because of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Table 8 on page 93 illustrates the tight clustering of allele sizes obtained on the 3500xL Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 nt The instance of a sample allele sizing outside of the 0 5 nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 nt or less Smith 1995 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra Peaks in the electropherogram For sample alleles that do not size within a 0 5 nt window the PCR product must be rerun to distinguish between a true off ladder allele vs measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment GeneMapper ID X Software automatically flags sample allele
85. eneAmp PCR System 9700 with the Silver 96 Well Block Veriti 96 Well Thermal Cycler Possible substrates for use with this kit include e Treated paper Copan NUCLEIC CARD System or Whatman FTA cards e Untreated paper 903 paper Bode Buccal DNA Collector Yfiler Plus PCR Amplification Kit User Guide 9 About the primers Loci amplified by the kit Overview Product overview Non nucleotide linkers are used in primer synthesis for the DYS3891 1L DYS635 DYS627 DYS19 YGATAH4 DYS448 DYS391 DYS390 DYS438 DYS391 DYS390 DYS438 DYS392 DYS518 DYS437 and DYS449 loci For these primers non nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis Butler 2005 Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus spacing The combination of a six dye fluorescent system and the inclusion of non nucleotide linkers allows for simultaneous amplification and efficient separation of the 27 Y STR loci during automated DNA fragment analysis The following table shows the loci amplified and the corresponding fluorescent marker dyes The Yfiler Plus Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the haplotype of the DNA Control 007 are also listed in the table Table 1 Yfiler Plus Kit
86. equence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 Most STR loci produce minus stutter peaks as a by product of PCR amplification A process of slippage has been proposed as a molecular mechanism for stutter where the Taq DNA polymerase enzyme slips on the template DNA during replication and produces a minority PCR product that is shorter than the template strand usually by one repeat unit The stutter process may also occur in the opposite direction to produce amplicon DNA that is usually one repeat unit longer than the template strand termed plus stutter While plus stutter is normally much less significant lt 5 than minus stutter in STR loci with tetranucleotide repeats the incidence of plus stutter is more significant in the trinucleotide repeat containing loci DYS481 and DYS392 as shown in Table 3 on page 71 GeneMapper ID X analysis files supplied for use with theYfiler Plus Kit contain plus stutter filters for several markers to prevent these peaks from being called in normal profiles To obtain a Technical Note regarding plus stutter in STR chemistries see your local Life Technologies HID support representative Yfiler Plus PCR Amplification Kit User Guide 69 70 Experiments and Results Extra Peaks in the electropherogram A non standard minus 2 nt stutter
87. equent genotyping e Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Yfiler Plus PCR Amplification Kit User Guide 45 4 Analyze Data Overview of GeneMapper ID X Software v1 4 e Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling e Alleles that are not in the Yfiler Plus Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol 46 Yfiler Plus PCR Amplification Kit User Guide Chapter 4 Analyze Data 4 Set up GeneMapper ID X Software for data analysis Set up GeneMapper ID X Software for data analysis Panel bin and The file names shown in this section may differ from the file names you see when you stutter file version download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Softw
88. erties The Quantifiler Human and Quantifiler Y Human Male Kits are highly specific for human DNA and they individually detect total human or male DNA respectively The kits detect single stranded and degraded DNA How they work The Quantifiler DNA Quantification Kits consist of target specific and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two locus specific PCR primers and one TagMan MGB probe labeled with FAM dye for detecting the amplified sequence The kits each contain a separate internal PCR control IPC assay which consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying the IPC template and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC Quantifiler Duo DNA Quantification Kit Part no 4387746 For more information see Quantifiler Duo DNA Quantification Kit User s Manual Part no 4391294 Properties The Quantifiler Duo Kit is highly specific for human DNA This kit combines the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two hu
89. f each component needed to prepare the reactions using the table below Reaction component Volume per reaction Primer Set 5 0 uL 10 0 uL Master Mix 10 0 uL PCR Low TE Buffer Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT This kit has been optimized for a 25 uL PCR volume to overcome the PCR inhibition expected when amplifying unpurified samples Using a lower PCR volume may reduce the ability of the kit chemistry to generate full STR profiles Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the reaction mix for 3 seconds then centrifuge briefly Dispense 25 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate The final volume in each well is 28 uL reaction mix plus Prep n Go Buffer and sample lysate or positive control Note For samples and controls add 3 uL of lysate or the 1 2 or 3 uL of control DNA in addition to the 25 uL of reaction mix There is no need to compensate for the volume of lysate control Add samples to the reaction plate Add the following to wells of a MicroAmp Optical Wells 96 Well Reaction Plate Test samples 3 uL of sample lysate Positive control For 25 and 26 cycles 3 uL of Control DNA 007 e For 27 cycles 2 uL of Control DNA 007 e For 28 c
90. g in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 Select Tools Panel Manager Check the version of files imported into the Panel Manager s Panel Manager File Edit Bins View Help Io y cy X a Select Panel Manager in the navigation pane Panel Manager Yfiler Plus PCR Amplification Kit User Guide 47 Analyze Data Set up GeneMapper ID X Software for data analysis b Expand the Panel Manager folder and any sub folders to identify the analysis file version already installed for your kit choice 4 Check the version of files available for import into the Panel Manager a Select Panel Manager then select File Import Panels to open the Import Panels dialog box b Navigate to then open the Panels folder and check the version of panel bin and stutter files installed 5 If newer versions are available on the website download and import as described below Import panels To import the latest Yfiler Plus Kit panel bin set and marker stutter files from our bins and marker web site into the GeneMapper ID X Software database stutter 1 Download and open the file containing panels bins and marker stutter a Go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software Download the file Yfiler Plus Analysis files_v1X
91. gative control reaction 23 positive control sample preparation 27 29 30 positive control reaction 23 quantification 20 quantification methods 20 sample preparation 23 test sample 23 using agarose gel analysis to examine the DNA 85 your sample preparation 27 DNA Polymerase catalyzing the addition of a3 A nucleotide 78 documentation related 123 DS 36 Matrix Standard 38 43 Dye Set J6 for 6 dye samples 38 43 E effect of inhibitors 84 electropherogram addition of a3 A nucleotide 78 electrophoresis Data Collection Software 35 41 prepare samples 38 43 references 35 41 run module 35 41 set up of 3130 3130xl instruments 41 set up of 3500 3500xL instruments 34 emission spectra 16 125 Index equipment not included with kit 109 F fluorescent dyes 15 FTA cards 26 G gels 85 gene diversity values 90 GeneMapper ID X Software analyze project 59 check version of panels bins and stutter 47 create analysis method 52 examine and edit project 60 import panels bins and stutter 48 overview 15 45 setup 47 GeneScan size standard about 17 dye label 15 volume per reaction 38 43 genetics allele frequencies 89 populations and samples used in studies 89 H HID Updater 36 Hi Di formamide volume per reaction 38 43 instrumentation 3130 3130xl genetic analyzer 15 3500 3500xL genetic analyzer 15 software compatibility 15 kit allelic ladder 17 amplification 9 contents 17 control DNA 17 description 9 f
92. he D21S11 locus Hum Mol Genet 1 67 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Sparkes R Kimpton C Watson S Oldroyd N Clayton T Barnett L Arnold J Thompson C Hale R Chapman J Urquhart A and Gill P 1996a The validation of a 7 locus multiplex STR test for use in forensic casework I Mixtures ageing degradation and species studies Int J Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996b The validation of a 7 locus multiplex STR test for use in forensic casework II Artifacts casework studies and success rates Int J Legal Med 109 195 204 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh P S 1998 SWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis J Forensic Sci 43 854 870 Wallin J M Holt C L Lazaruk K D Nguyen T H and Walsh P S 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Yfiler Plus PCR Amplification Kit User Guide 121 Bibliography 122 Yfiler Plus PCR Amplification Kit User Guide Documen
93. ied with this kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Do not refreeze kit components after thawing The following table lists data collection software and the run modules that can be used to analyze PCR products generated by this kit For details on the procedures refer to the documents listed in the table reference documents i Data a gene Operating Collection Additonal Run modules and conditions References Analyzer System software Software 3130 or Windows Data 3130 3730 DC HIDFragmentAnalysis36_POP4_1 Applied Biosystems 3130xff 7 Collection v4 6 Dye Injection conditions for 3130 3130 Series Data Software Module v1 3 kV 5 sec Collection Software v4 v4 contact Life Getting Started Guide Technologies Injection conditions for 3130xl 3 kV 10 sec Alternate injection conditions for the 3130xl 3 kV 13 sect Run conditions 15 kV 1500 sec a Dye Set J6 Pub no 4477796 We conducted validation studies for the Yfiler Plus Kit using the 3130xl 3500 or 3500xL configurations This kit was developed using two injection voltage conditions for the 3130xl 3 kV 10 sec and 3 kV 13 sec You are encouraged to explore both options during validation to determine which protocol provides the best results on your instrumentation Obtain a
94. ill not be imported into the GeneMapper ID X Software database Yfiler Plus PCR Amplification Kit User Guide 51 Analyze Data Set up GeneMapper ID X Software for data analysis Create an analysis Use the following procedure to create an analysis method for the Yfiler Plus Kit method IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For example an analysis method created for GeneMapper ID X version 1 2 is not compatible with earlier versions of GeneMapper ID X Software or with GeneMapper ID Software version 3 2 1 1 Select Tools gt GeneMapper ID X Manager to open the GeneMapper ID X Manager GeneMapper ID X Manager Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings 2 Select the Analysis Methods tab then click New to open the Analysis Method Editor with the General tab selected The figures below show the settings for each tab of the Analysis Method Editor Configure the Analysis Method Editor tab settings as shown in the figures below unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 3 After you enter settings in all tabs click Save 52 Yfiler Plus PCR Amplifica
95. ion Use recommended amount of template DNA 1 0 ng Incomplete denaturation of double stranded DNA Use recommended amount of Hi Di Formamide and perform heat denaturation step according to the instructions in Perform electrophoresis on page 33 Some but not all loci visible on electropherogram of DNA Test Samples Less than 25 uL of PCR volume was used Repeat amplification using the recommended PCR volume of 25 jL 106 Yfiler Plus PCR Amplification Kit User Guide Appendix BTroubleshooting E Observation Possible causes Recommended actions STR profiles contain many off scale alleles DNA quantitation was not performed or not accurate Verify the accuracy of the DNA quantitation protocol Poor peak height balance Incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters Yfiler Plus PCR Amplification Kit User Guide 107 El Troubleshooting 108 Yfiler Plus PCR Amplification Kit User Guide C Ordering information Equipment and materials not included Table 10 Equipment Equipment Source 3500 3500xL Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer for Human Identification Applied Biosystems 3130 3130x Genetic Analyzer Contact your local Life Technologies sales representative Veriti 96 Well Thermal Cycler 4479071 GeneAmp PCR System
96. ion during thermal cycling The Veriti Thermal Cycler does not require a compression pad 3 Start the run 4 On completion of the run store the amplified DNA and protect from light If you are storing the DNA Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C IMPORTANT Store the amplified products so that they are protected from light 32 Yfiler Plus PCR Amplification Kit User Guide Perform electrophoresis Allelic ladder requirements KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK K 33 m Section 3 1 3500 3500xL instruments ooo 34 Set up the 3500 3500xL instruments for electrophoresis k3 34 Prepare samples for electrophoresis on the 3500 3500xL instruments 38 Section 3 2 3130 3130xl instruments eee 41 Set up the 3130 3130xl instruments for electrophoresis nn 41 Prepare samples for electrophoresis on the 3130 3130xl instruments 43 Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples Number of One e ene Number of samples per allelic Instrument allelic ladders injection ladder s to run equals 3500 1 per 3injections 8 samples 23 samples 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples 1 allelic ladder 3130 1 per 4 injections 4 samples 15 samples 1 allelic ladder 3130xl 1 per injection 16 samples 15 samples 1 allelic ladder
97. ion pane a Panel Manager dil File Edit Bins View Help wx al sm BIE onset tepel GE E sE Panel Manager Panel Hame Comment GamprisTR Panels vak 1 none EN mt Y filer _Plus_v1 Cok cone Ac L Heb b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the Yfiler_Plus_Panels_v1 folder d Select Yfiler_Plus_Bins_v1 txt then click Import Note Importing this file associates the bin set with the panels in the Yfiler Plus Panels v1 folder T E Yfiler_Plus Bins_v1 tet ES Yfiler_Plus_Panels_v1 txt Recent Items j Yfiler_Plus_Stutter_v1 t File name Yfiler_Plus_Bins_v1 Files of type all Files A Yfiler Plus PCR Amplification Kit User Guide 49 Analyze Data Set up GeneMapper ID X Software for data analysis 7 To view the imported panels in the navigation pane double click the Yfiler_Plus_Panels_v1 folder to display the panel information in the right pane ww Panel Manager File Edit Bins View Help ui gt a S aa Panel Manager Marker Name Dye Color Max Size Control Alleles Ladder Alleles u u u u u u o AmpFLSTR_Panels_v3X SS76 Blue 138 0 19 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Yfiler_Plus_v1 DYS3891 Blue 184 0 13 9 10 11 12 13 14 15 16 17 EY fier _Plus_Panel_v 1 5 DYfler_Plus Panel vil gt yeess
98. iosystems 3500xL Genetic Analyzer Figure 4 The performance of the multiplex is most robust within 20 of the optimal magnesium chloride concentration Figure 4 Amplification of a mixture of 1 ng of male 007 DNA and 1 ug of female 9947 DNA with varying concentrations of MgCl analyzed on the 3500xL Genetic Analyzer Y axis scale is 0 to 13 000 RFU Elmark Same for Deletion 20 i ka t a L La L irla uli j i ji i 10 4 pta a di diirid li Ad j Optimal yla i dla lll ly Aili Lil i pita da tidus lult j a i i j i at 20 Yfiler Plus PCR Amplification Kit User Guide Thermal cycler parameters Chapter 5 Experiments and Results Developmental validation Figure 5 Electropherograms obtained from amplification of a blood sample on FTA card amplified with the Yfiler Plus Kit in the presence of varying concentrations of magnesium chloride and analyzed on an Applied Biosystems 3500xL Genetic Analyzer Y axis scale 0 to 10 000 RFU TI Merk Sample for Deleon 20 ET Nn dala 10 gl La Lb h La li data Optimal aia aa iL l Ll Aya li Jalad j 7 NERA SEN 10 NE 20 a a dil ii di Ir ia u a iial Figure 6 Electropherograms obtained from amplification of a buccal sample on FTA card amplified with the Yfiler Plus Kit in the presence of varying conce
99. kK KK kk 67 SWGDAM Guideline 3 5 soes 67 ACEUPACY ss UA ee ee etd UE LEENE EEA EAA ARE 67 Precision and size windows nnen ennen ennen 68 Extra Peaks in the electropherogram eee eeen 69 Causes of extra peaks sns sor ann ene san ae diwani hee ikak dk Leet nde 69 Stutterproduets snaren der re gerede Mee eee ag ae deed oe Mee ees 69 Addition of 3 Ancene saanderrere nk verd etek ete wade tene edn ah 78 Abo bartilactS mr oie aches EEE Ze E diha ee Le 79 Other DNA dependent artifacts Ml oee 79 Characterization Of lOGI anus vernemen eee BRE Pens BEEN deelt Av dn RER Qe Rd 81 SWGDAM Guideline 3 1 sonen eenen 81 Nature of the polymorphisms eee 81 Nal ald elit otc N VD Nae eee AE RENE 81 VIET oT ae EN 81 Speci s specificity en arn nn Pes dco da peed cose ERE NRNRNRENnNuNNDERZRMN 82 SWGDAM Guideline 3 2 noen 82 Sh NY VAN DD DD ende eN tan eee bg 83 SWGDAM Guideline 3 3 sense 83 Effect of DNA quantity on results and importance of quantitation 83 Stability 5 2 eaten nna tend B aE Satinne doe T Vou eens dau datas O tienes 84 SWGDAM Guideline 3 4 ooo eeen kk kk kk kk kk kk kk kk kk kk kk kk kk kk ees 84 Lack of amplification of Some loci kk kk kk kk kk kk kK kK KK KK KK KK KK KK KK kK KK kk k 84 Effecto inhiDItO RS ks Aa dana ae e na hee ese ret A KL en 84 Degraded DNA R Aiel Ni et tee ihe Gh Mec tothe seh beren la DM A 85 Mixture Studi s 20 0 5 22 wenn ame eee eee eset Co eed pee Vl HH mm 87 SWGDAM Guideline
100. l DNA 007 in the presence of female DNA 9947A Profiles shown in the panels from top to bottom 1 ng of male DNA 1 ng male DNA with 1 ug female DNA 500 pg male DNA with 1 ug female DNA 250 pg male DNA with 1 ug female DNA 1 ug female DNA Note that the Y axis scale is magnified for lower input amounts of male DNA samples which generate lower peak heights Y axis scale is 0 to 200 RFUs for the 1 ug female input 20000 Tur iin a a Mark Sample for Deletion 2000 a L i ti dak H l J Mark Sample for Deletion Wo ii i la ti lili sili an Mark Semple for Deletion EJ RE Aidi zen 260 1 4000 Mark Sample for Deletion Tug female Yfiler Plus PCR Amplification Kit User Guide 87 5 Experiments and Results Mixture studies Male male mixture Forensic samples may contain body fluids or tissues originating from more than one studies male Mixtures of two male DNA samples were examined at various ratios 1 1 to 1 15 The total amount of genomic input DNA mixed at each ratio was 1 ng Table 5 Haplotypes of samples in Figure 26 Allele Sample A Sample B DYS576 15 19 DYS389 14 13 DYS635 21 24 DYS389II 31 29 DYS627 21 21 DYS460 10 11 DYS458 17 17 DYS19 15 15 YGATAH4 12 13 DYS448 21 19 DYS391 10 11 DYS456 13 15 DYS390 21 24 DYS438 12 12 DYS392 11 13 DYS518 38 37 D
101. lelic dropout and minimal occurrence of off scale allele peaks Instrument Peak height 31xx 2500 4000 RFU 3500 Series 5000 12 000 RFU Treated or untreated paper prepare reactions Sample prep Do not add water to the wells on the reaction plate before adding the punches If guidelines your laboratory is experiencing static issues with the paper discs you may prepare and dispense the 25 uL reaction mix into the wells of the reaction plate before adding the punches e For manual punching Place the tip of a 1 2 mm Harris Micro Punch on the card hold the barrel of the Harris Micro Punch do not touch the plunger gently press and twist 1 4 turn then eject the punch into the appropriate well on the reaction plate e For automated punching Please refer to the User Guide of your automated or semi automated disc punch instrument for proper guidance e For blood on untreated paper samples add 2 uL of Prep n Go buffer on top of the 1 2 mm sample punch 26 Yfiler Plus PCR Amplification Kit User Guide Section 2 2 Direct amplification of DNA Treated or untreated paper prepare reactions Prepa re the 1 Add samples to the reaction plate reactions well s Add the following to wells of a MicroAmp Optical 96 Well Reaction Plate Negative control 1 2 mm blank disc Test samples 1 2 mm sample disc Positive control For 26 cycles 3 uL of Control DNA 007 IMPORTANT Do not add a For 27 cycles
102. ll i u al dd j l l 61 C sdi kf li A ul ly ijl i al F i 61 5 C diit W dik had s ul x i 62 C NE l iil i li z ij i ji 62 5 C aa a lk al Ju Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Accuracy precision and reproducibility Figure 9 Electropherograms obtained from amplification of a buccal sample on an FTA card at annealing temperatures of 60 5 61 61 5 62 and 62 5 C analyzed on an Applied Biosystems 3500xL Genetic Analyzer Y axis scale is 0 to 10 000 RFU F Merk Sample fer Deletar ar ji j 60 5 C Alet ul ii i dl hl lii e a B n EJ E La 61 C Jiii tl iri hi iini roe F l l l j l 1 61 5 C Lii LL lil u jl wi Ada Ll i Li LL li li d Wa ddu dal al 7 7 gt 62 5 C al ee tae 1 Lu L Ai i di ul kj a L_l al Accuracy precision and reproducibility SWGDAM Guideline 3 5 Accuracy Precision and accuracy of the assay should be demonstrated Precision characterizes the degree of mutual agreement among a series of individual measurements values and or results Precision depends only on the distribution of random errors and does not relate to the true value or specified value The measure of precision is usually expressed in terms of imprecision and computed as a standard deviation of the te
103. luorescent dyes 15 loci amplification 10 PCR reaction mix 17 22 primers 10 17 21 126 reagents 17 supported instruments 9 thermal cyclers for use with 114 L license activation 6 dye 41 limited product warranty 124 LIZ size standard about 17 volume per reaction 38 43 loci allele frequencies in the population databases 89 differential amplification 85 lack of amplification effect of DNA quantity on results 83 population data allele frequencies 89 population data samples used in studies 89 low TE buffer 20 lysate prepare 28 M master mix volume per reaction 27 master mix volume per reaction 30 materials and equipment included in kit 17 mixed samples 87 multicomponent analysis 15 N negative control sample preparation 23 Nucleic Card System 9 0 operating systems 15 35 41 P panel import 48 panels check version 47 PCR optimize cycle number 25 performing 23 31 setup 113 Yfiler Plus PCR Amplification Kit User Guide thermal cycling conditions programming 23 31 PCR work areas 109 113 population genetics allele frequencies 89 populations and samples used in the studies 89 positive control sample preparation 23 Prep n Go Buffer 25 primers volume per reaction 27 30 primers volume per reaction 22 punches size 26 Q quantification DNA 20 R reaction mix for PCR 27 30 reaction mix volume per reaction 22 reactions prepare for PCR 29 reactions preparing for PCR 22 reagent
104. lus PCR Amplification Kit User Guide Appendix ATable of Precision Results 3130xl 3500 3500xL Allele Standard Standard Standard Mean deviation Mean deviation mean deviation 10 177 93 177 98 0 027 0 035 178 26 178 28 0 031 0 053 178 20 178 22 0 030 0 047 11 181 97 182 01 0 026 0 036 182 29 182 32 0 030 0 045 182 24 182 26 0 040 0 046 12 186 02 186 07 0 021 0 041 186 35 186 38 0 027 0 041 186 31 186 32 0 031 0 046 13 189 98 190 02 0 020 0 039 190 28 190 31 0 020 0 051 190 24 190 26 0 033 0 043 14 194 03 194 07 0 023 0 041 194 34 194 37 0 028 0 049 194 29 194 31 0 041 0 044 15 198 15 198 20 0 025 0 031 198 49 198 51 0 017 0 040 198 46 198 47 0 035 0 046 16 202 13 202 19 0 030 0 037 202 47 202 50 0 026 0 047 202 44 202 45 0 035 0 041 17 206 08 206 12 0 023 0 034 206 42 206 45 0 026 0 056 206 36 206 38 0 033 0 040 18 210 03 210 07 0 022 0 036 210 38 210 41 0 031 0 054 210 33 210 35 0 038 0 045 DYS438 6 207 14 207 22 0 024 0 032 207 69 207 73 0 044 0 059 207 67 207 68 0 033 0 040 7 212 16 212 22 0 025 0 032 212 70 212 71 0 030 0 049 212 66 212 68 0 030 0 054 8 217 23 217 31 0 022 0 038 217 78 217
105. man specific assays in one PCR reaction for total human DNA and human male DNA The two human DNA specific assays each consist of two PCR primers and a TaqMan probe The TaqgMan probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye Quantifiler HP DNA Quantification Kit Cat no 4482911 For more information see Quantifiler HP and Trio DNA Quantification Kits User Guide Pub no 4485354 Properties The Quantifiler HP Kit is designed to quantify the total amount of amplifiable human DNA in a sample How it works The Quantifiler HP DNA Quantification Kit uses multiple copy target loci for improved detection sensitivity The human specific target loci Small Autosomal Large Autosomal and Y chromosome targets each consist of multiple copies dispersed on various autosomal chromosomes Small Autosomal and Large Autosomal To maximize the consistency of quantification results genomic targets were selected with conserved primer and probe binding sites within individual genomes and also with minimal copy number variability between different individuals and population groups As a result the detection sensitivity of the assay is improved over Quantifiler Duo Human and Y Human Male DNA Quantification Kit ass
106. mation about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Yfiler Plus PCR Amplification Kit User Guide 117 E Safety Biological hazard safety 118 Yfiler Plus PCR Amplification Kit User Guide Bibliography Ballantyne K N Goedbloed M Fang R et al 2010 Mutability of Y chronosomal microsatellites rates characteristics molecular bases and forensic implications Am J Hum Genet 87 341 353 Ballantyne K N Keerl V Wollstein A Choi Y Zuniga S B Ralf A Vermeulen M de Knijff P Kayser M 2012 A new future of forensic Y chromosome analysis rapidly mutating Y STRs for differentiating male relatives and paternal lineages Forensic Sci Int Genet 6 2 208 18 Ballantyne K N Ralf A Aboukhalid R et al 2014 Towards male individualization with rapidly mutating Y chromosomal STRs Hum Mutat doi 10 1002 humu 22599 PubMed PMID 24917567 Begovich A B McClure G R Suraj V C Helmuth R C Fildes N Bugawan T L Erlich H A and Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J Immunol 148 249 58 Butler J M 2005 Forensic D
107. n the Y chromosome Due to the lack of recombination the entire Y chromosome haplotype must be treated as a single locus Haplotype frequencies are estimated using the counting method The counting method involves searching a given haplotype against a database to determine the number of times the haplotype was observed in that database The frequency of the haplotype in the database is then estimated by dividing the count by the number of haplotypes searched SWGDAM January 2014 The Yfiler Plus Kit was used to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference Population Number of samples African American 557 U S Caucasian 533 U S Hispanic 391 U S Asian 340 Table 6 shows the Yfiler Plus Kit gene diversity in three populations listed as percentages Yfiler Plus PCR Amplification Kit User Guide 89 Experiments and Results Population data Table 6 Yfiler Plus Kit Gene Diversity values across four different U S populations Locus heen Ba Hank ker do nl n 557 n 533 ren Lara DYS576 0 807 0 768 0 769 0 799 DYS389 0 504 0 527 0 567 0 679 DYS635 0 716 0 646 0 713 0 786 DYS389II 0 746 0 676 0 729 0 770 DYS627 0 838 0 842 0 853 0 812 DYS460 0 573 0 537 0 571 0 675 DYS458 0 750 0 766 0 800 0 820 DYS19
108. n the literature Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 The Yfiler Plus Kit results confirmed that the loci were inherited according to a Y linked father to son transmission In no case was the maternal grandfather s Y haplotype found in the offspring Calculation of a mutation rate based on this small population size would be inaccurate due to the small sample size The samples were reamplified and reinjected to confirm the allele call The Yfiler Plus Kit loci have been mapped and the chromosomal location on the Y chromosome is known based on the nucleotide sequence of the Y chromosome The Genbank accession numbers for representative sequences are DYS19 X77751 AC017019 DYS385 AC022486 Z93950 DYS389 AC011289 AF140635 DYS390 AC011289 DYS391 G09613 ACO11302 DYS392 G09867 AC06152 DYS393 G09601 AC06152 DYS437 AC002992 DYS438 AC002531 DYS439 AC002992 Yfiler Plus PCR Amplification Kit User Guide 81 5 Experiments and Results Species specificity DYS448 AC025227 6 DYS456 AC010106 2 DYS458 AC010902 4 DYS635 G42676 AC011751 DYS635 G42673 DYS449 AC051663 DYS481 FJ828747 1 DYS533 AC053516 DYS570 AC012068 DYS576 AC010104 DYS518 FJ828760 and DYS627 BV208976 Species specificity SWGDAM Guideline 3 2 82 The ability to detect genetic information from non targeted sp
109. nd activate the 6 dye license for the instrument 1 Confirm that you are running Data Collection Software v4 Help gt About 2 Obtain a 3130 DC v4 6 Dye Module v1 License key Contact Life Technologies for information 3 Ensure that all network cards in the computer are enabled IMPORTANT You can run the 3130 Series Data Collection Software v4 using only the network cards enabled when you activate the software license For example if you activate the software when your wireless network card is disabled you will not be able to run the software when the wireless network card is enabled Yfiler Plus PCR Amplification Kit User Guide 41 Set up the 3130 3130xl instruments for electrophoresis 4 Select Tools License Manager to display the Software Activation dialog box 3xxx Series Data Collection Software 4 Software Activation a 1 Request license file for Computer ID 002564ee 13a4 002564ee13a5 This ID is unique to this computer and cannot be used to obtain a license file for another computer a Enter the license key from CD or email AID 166c Saaf 030c 462e a163 974c e6c7 12a6 b Enter your email address john doe lifetech com c Is this computer currently connected to the internet Yes Connected No Not Connected 2 Retrieve the license file from email then save it to the desktop of this computer 3 Find the license file 4 Click Install and Validate License
110. ng the Veriti 96 Well Thermal Cycler refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation Part no 4440754 Initial incubation step Denature Anneal Final Final hold Extend extension HOLD CYCLE 30 HOLD HOLD 95 C 94 C 61 5 C 60 C 4 C oo 1 min 4 sec 1min 22 min Load the plate into the thermal cycler and close the heated cover IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad Start the run On completion of the run store the amplified DNA and protect from light Yfiler Plus PCR Amplification Kit User Guide 23 2 Perform PCR Perform PCR If you are storing the DNA Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C IMPORTANT Store the amplified products so that they are protected from light 24 Yfiler Plus PCR Amplification Kit User Guide Section 2 2 Direct amplification of DNA 2 Optimize PCR cycle number Section 2 2 Direct amplification of DNA Optimize PCR cycle number Select samples and prepare plates Before
111. nhibitors 84 Figure 22 Effect of amplifying 1 ng 500 pg 250 pg 125 pg 62 pg and 31 pg of male control DNA 007 using two voltage conditions Data analyzed using the 3500xL Genetic Analyzer The Y axis scale is 0 to 12 000 RFUs 12000 Voltage 1 2 kv 10000 J do n e 8000 e 6000 1 a 4000 a e 2000 r e s 04 ing 500pg 250pg 125pg 62 5pg 31 25pg The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected to various environmental and chemical insults should be evaluated In most instances assessment of the effects of these factors on new forensic DNA procedures is not required However if substrates and or environmental and or chemical insults could potentially affect the analytical process then the process should be evaluated to determine the effects of such factors SWGDAM December 2012 As with any multi locus system the possibility exists that not every locus will amplify This is most often observed when the DNA sample contains PCR inhibitors or when the DNA sample has been severely degraded Valuable information may be obtained from partial profiles Traces of humic acid may inhibit the PCR amplification of DNA evidence collected from soil Amplification of 1 0 ng of DNA Control 007 in the presence of increasing amounts of humic acid was performed using the Yfiler Plus Kit Figure 23 on page 8
112. ntrations of magnesium chloride and analyzed on an Applied Biosystems 3500xL Genetic Analyzer Y axis scale 0 to 28 000 RFU T Mark Sarpi for Deletior a al al la TJ Nark Serpe for Deletier 10 boy ba tut ron mark Sare for Deer he jili ak San for Deletar l_j l iii Wy Li li liil lii Thermal cycling parameters were established for amplification of the Yfiler Plus Kit in the Veriti 96 Well Thermal Cycler Thermal cycling times and temperatures of GeneAmp PCR systems were verified Annealing and denaturation temperature windows were tested around each stipend to verify that a 1 09C window produced a specific PCR product with the desired specificity for male DNA Optimal 20 The effects of denaturation and annealing temperatures on the amplification of Yfiler Plus Kit loci were examined using the control DNA 007 male female DNA mixtures blood FTA and buccal FTA samples Yfiler Plus PCR Amplification Kit User Guide 65 66 Experiments and Results Developmental validation The denaturation temperatures tested were 93 94 and 95 C all for 4 second hold times on the Veriti 96 Well Thermal Cycler The annealing temperatures tested were 60 5 61 61 5 62 and 62 5 C Figures 7 8 and 9 for 1 minute hold times in the Veriti 96 Well Thermal Cycler The PCR products were analyzed using the 3500xL Genetic Analyzer No preferen
113. number ir miira Wa r ka ba ee D WAE WT WEN a Ar 24 2 0 25 Select samples and prepare plates kk kk kk kk kk kK KK KK kK KK KK KK kK kk KK kk k 25 Determine optimum conditions WWW kk kk kk kk kk kk kk kk kK kk KK kk kk kk kk kk kK kk kk kk kk lk 26 Treated or untreated paper prepare reactions kk kk kk KK kK KK KK KK KK KK KK KK KK 26 Yfiler Plus PCR Amplification Kit User Guide 3 Contents sample prep Guidelines y tor al 21 14y ce ale ie erneer lan Sach kala en 26 Prepare the reactions sens cee teat Sag ee aen deter terne netten dta Ahern ad 27 Swab substrates prepare reactions WWW kk kk kk kk kk kk kk kK KK KK kK KK kk kk kk kk kk kk kk kk kk kk 28 Sample prep guidelines kk kk 0c cece cece kk kk kk kk kk kk kk kk kk kk kk kk kk ka 28 Prepare the sample lysate room temperature protocol WWW KRA 28 Prepare the sample lysate heat protocol eeen 28 Prepare the reactions A key ek bandera inr Weens Hendrie ede dg 29 Store the sample lysate gt k noon eneen eenen eeen 31 Perform POR nen vaneen SE a ka fd Wd A en ne wad 31 CHAPTER 3 Perform electrophoresis nnen 33 Allelic ladder requirements eneen eene eneen een 33 Section 3 1 3500 3500xL instruments nenten een a neet tn taeda ts 34 Set up the 3500 3500xL instruments for electrophoresis onee KK KK KK 34 Reagentsandiparts ss ser mannen vane Ee vd Be EE EER Edd 34 Electrophoresis software setup and reference documents oon 35 Ob
114. o 4484678 This kit contains enough reagents for two sets of 50 reactions 500 reactions Cat no 4482730 This kit contains enough reagents for two sets of 250 reactions IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Component Description 100 reactions 500 reactions Storage Yfiler Plus Contains enzyme salts dNTPs 2 tubes 4 tubes 15 to 25 C on receipt Master Mix bovine serum albumin and 0 05 0 5 mL tube 1 25 mL tube 2 to 8 C after initial use sodium azide in buffer and salt DNA Contains 2 0 ng L human male 1 tube 0 05 mL 2 tubes Control 007 genomic DNA in 0 05 sodium 0 05 mL tube azide and buffert See Table 1 on page 10 for profile Yfiler Plus Contains locus specific dye 2 tubes 2 tubes 15 to 25 C on receipt Primer Set labeled and unlabeled forwardand 0 25 mL tube 1 25 mL tube 2 to 8 C after initial use reverse primers to amplify human Store protected from male DNA target light Yfiler Plus Contains amplified alleles 2 tubes 2 tubes Allelic Ladder See Table 1 on page 10 for a list of 0 025 mL tube 0 05 mL tube alleles included in the allelic ladder The DNA Control 007 is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of
115. ofile zot si bal d hedd ee ek layan Pele hs 12 Workflow overview for casework samples kk kk kk kk kk kK kk ee kK kk kk kk kk kk kk kk k 13 Workflow overview for database samples WWW kk kk kk kk kk kk kK kK kk KK KK KK KK kK kk KK kk k 14 Instrument and software overview WWW kk kk kk kk kk kk kK kK KK kk kk kk kk kk kk kk kk kk kk kk kk lk kk 15 Data Collection and GeneMapper D X Software kk kK KK KK KK KK KK KK KK KI 15 Instrument and software compatibility oen 15 About multicomponent analysis kk kk kk oo eee eee 15 How multicomponent analysis works oon eee 15 Materials and equipment kk kk kk kk kk kk kk kK kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk 17 Kit contents and storage WW nnen ennen kK KK kk kk kk kk kk kk kk kk kk kk eae 17 Standards for samples n n n nannan urrann nnr kk kK kk kk kK kk kk kk kk kk kk kk kk kk kk kk k 17 B CHAPTER 2 Perform PCR lt JL kk kk kk kk kk kk kk kk kk ene daama 19 Section 2 1 Amplification from extracted DNA nnn 20 Required user supplied reagents eenen eeen 20 DNAvGuANTITICAtION es sar dlee nternet a eeste edt n HH HH gt E 20 Importance of quantification nennen 20 Methods of quantifying DNA 0c kk kk kk kk kK kk kk kk kk kk kk kk kk kk kk kk kk kk 20 Prepare the amplification kit reactions eeen 22 Perton GR ten Ne Ee NEN cer tet tee An 202 adhd een nde 23 Section 2 2 Direct amplification of DNA ene 25 Optimize PGRicycle
116. ogram Figure 16 Minus stutter percentages for the DYS533 DYS570 and DYS576 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively Percent Stutter 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 mese ve o bd ezmane e ee o 6 7 8 9 10 1112 13 14 15 16 17 18 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 DYS533 DYS570 DYS576 Figure 17 Minus stutter percentages for the DYS627 DYS635 and YGATAH4 loci Blue green black red and purple colors indicate loci labeled with 6 FAM VIC NED TAZ and SID dyes respectively Percent Stutter 30 0 29 0 28 0 27 0 26 0 25 0 24 0 23 0 22 0 21 0 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 commen o cmm ose o o cms o oe ecm some eamene memmen vanme emme e e comme lt e e wee 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 DYS627 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 7 8 9 10 1112 13 14 15 16 DYS635 YGATAH4 Yfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Extra Peaks in the electropherogram Figure 18 Example of reproducible 2 nt stutters in the DYS19 lef
117. omplete electrophoresis using a single 96 well plate thus minimizing the impact of run to run variation on the results 2 Prepare the samples and the reactions as described in the protocols later in this chapter Prepare sufficient PCR reagents to complete amplification of three replicate plates 3 Create three identical PCR plates Yfiler Plus PCR Amplification Kit User Guide 25 Treated or untreated paper prepare reactions 4 Amplify each plate using a different cycle number to determine the optimum conditions for use in your laboratory Suggested cycle numbers for different sample type and substrate combinations are listed below Substrate Sample type Treated or untreated paper Blood 26 27 28 29 cycles Buccal 26 27 28 29 cycles Note To minimize the effect of instrument to instrument variation use the same thermal cycler to amplify all three plates To maximize result quality prepare and amplify Plate 1 then repeat for Plates 2 and 3 Do not prepare all three plates simultaneously Determine 1 Run the PCR products on the appropriate CE platform using the recommended optimum protocol see Chapter 3 Perform electrophoresis on page 33 conditions 2 Based on the results of the sensitivity study select the appropriate PCR cycle number for future experiments Our studies indicate the optimum PCR cycle number should generate profiles with the following peak heights with no instances of al
118. or its latest version b Unzip the file 2 Start the GeneMapper ID X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 3 Select Tools Panel Manager 4 Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane b Select File Import Panels to open the Import s Panel Manager Panels dialog box File Edit Bins View Hi c Navigate to then open the Yfiler Plus Analysis files_v1X folder that you unzipped in step 1 on page 48 48 Yfiler Plus PCR Amplification Kit User Guide Chapter 4 Analyze Data 4 Set up GeneMapper ID X Software for data analysis 5 Select Yfiler_Plus_Panels_v1 txt or the version you installed then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager Yfiler_Plus_Panels_v1 This folder contains the panel for the Yfiler Plus Kit and associated markers u Import Panels Look in Fm Yfiler Plus Analysis files_v1X PERE A L Vfiler_ Plus Bins _vl tdt j 2 Yfiler_Plus_Panels_v1 txt Recent Items L Yfiler_Plus_Stutter_v1 tet File name Yfiler _Plus_Panels_v1 txt Import Cancel Files of type ail Files 6 Import Yfiler_Plus_Bins_v1 txt a Select the Yfiler_Plus_Panels_v1 folder in the navigat
119. osol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Vortex MLS t For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Yfiler Plus PCR Amplification Kit User Guide 111 Ordering information Equipment and materials not included 112 Yfiler Plus PCR Amplification Kit User Guide PCR Work Areas Work area setup and lab design 0 6 6 6 KK KK KK KK KK KK KK KK KK KK KK KK 113 PCR setup work area n k s Wale E ba bana AER eee 113 Amplified DNA work area W K KK KK KK KK KK KK KK KK KK KK KK KK KK KK 114 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory If you are using the Yfiler Plus PCR Amplification Kit for e Forensic DNA testing refer to Forensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 e Parentage DNA testing refer to the Guidance for Standards for Parentage Relationship Testing Laboratories American Association of Blood Banks 7th edition 2004 The sensitivity of the Yfiler Plus Kit and other PCR based tests enables amplification of minute quanti
120. p Table Setting 3500 Data Analysis 6Dye i gt amp z Bi amp Analysis Completed Samples Analysis Summary Genotypes Analysis Summary Summary Generation Date Jun 24 2014 3 58 5 Select run folder to display All Sample Status JC Total of Samples Unanalyzed 0 Analyzed a UR Analysis Setting Changed EJ Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folder based on SQ and CGQ only Run Folder Total of Analyzed Ladders J Inj5 2014 04 19 09 44 42 917 1 Inj6 2014 04 19 09 44 42 927 T Bae 1 Control Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Control Type _ Total of Samples _ lj Al thresholds met ___ One or more thresholds not met Positive Control 0 Custom Control Negative Control Total ji o La o 0 0 o ololole Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Total of samples _ B Al thresholds met One or more thresholds not met Samples 34 21 3 Examine and edit a project 60 You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Analysis Summary tab of the Project windo
121. p the 3500 3500xL instruments for electrophoresis the documents listed in the table The following table lists data collection software and the run modules that you can use to analyze PCR products generated by this kit For details on the procedures refer to reference documents Genetic Operating pala Additional Plate templates assays pun Anayz r Svetem Collection software modules and conditions References y y Software installed with the HID Updater 35008 Windows 3500 Data HID Updater Plate templates 6dye_36_POP4 3500 3500xL Genetic Vista Collection 3500 DC e Assays GF Norm_POP4 and Analyzer User Guide Software v2 0 GF_POP4 which contain instrument Pub no 4401661 v2 0 Part no protocol HID36_POP4_J6_NT3200 HID Updater 3500 Data 4480670 with the following conditions Collection Software v2 Run module HID36 POP4 Release Notes Injection conditions 1 2 kV 16 sect Alternate injection conditions 1 5 kV 16 sect Run conditions 13 kV 1550 sec Dye Set J6 3500xL8 e Plate templates 6dye_36_POP4_xl e Assays GF Norm_POP4_xl and GF_POP4_xl which contain instrument protocol HID36_POP4xl_J6_NT3200 with the following conditions Run module HID36_POP4xl Injection conditions 1 2 kV 24 sec Alternate injection conditions 1 5 kV 24 sectt Run conditions 13 kV 1550 sec Dye Set J6 35008 Windows 7 3500 Data HID Updater Same as 3500 Data Collection Software 3500 3500xL Geneti
122. propriate amount of size standard based on your experiments and results Pipet the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate add e 10 uL of the formamide size standard mixture e 1 ul of PCR product or Allelic Ladder Note For blank wells add 10 uL of Hi Di Formamide Yfiler Plus PCR Amplification Kit User Guide Section 3 1 3500 3500xL instruments Prepare samples for electrophoresis on the 3500 3500xL instruments 5 Seal the reaction plate with appropriate septa then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Place the plate assembly on the autosampler Oreel GON Start the electrophoresis run Yfiler Plus PCR Amplification Kit User Guide 39 3 Perform electrophoresis Prepare samples for electrophoresis on the 3500 3500xL instruments 40 Yfiler Plus PCR Amplification Kit User Guide Set up the 3130 3130xl instruments for electrophoresis Section 3 2 3130 3130xl instruments Set up the 3130 3130xl instruments for electrophoresis Reagents and parts Electrophoresis software setup and Appendix C Ordering information on page 109 lists the required materials not suppl
123. roAmp Clear Adhesive Film Store the sample lysate as needed If you are storing the sample lysate Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C These storage recommendations are preliminary pending the results of ongoing stability studies The effects of multiple freeze thaw cycles on the lysate have not been fully evaluated Therefore multiple freeze thaw cycles are not recommended Perform PCR 1 Program the thermal cycling conditions When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode When using the Veriti 96 Well Thermal Cycler refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation Part no 4440754 Initial incubation Denature Anneal Extend Final Final hold step Extension HOLD CYCLE 26 29 HOLD HOLD 95 C 9496 61 5 C 60 09C 4 C 1 min 4 sec 1 min 22 min co Yfiler Plus PCR Amplification Kit User Guide 31 Perform PCR 2 Load the plate into the thermal cycler and close the heated cover IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad Cat no 4312639 on top of the plate to prevent evaporat
124. road Peak BD 0 5 Allelic Ladder GQ Weighting Spike SSPK SPK i Off scale OS lv SQ amp GQ Ranges PassRange Ren Sizing Quality From 0 75 to 1 0 From 0 0 to 0 25 Genotype Quality From 0 75 to 1 0 From 0 0 to 0 25 Reset Defaults SaveAs save Cancel Help IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform appropriate internal validation studies to determine the appropriate values to use The GS600_LIZ_ 60 460 size standard definition provided with GeneMapper ID X Software v1 4 and used with the Local Southern size calling method contains the following peaks GeneScan 600 LIZ Size Standard v2 0 60 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 400 414 420 440 and 460 Note This size standard definition has been validated for use with the Yfiler Plus Kit on the Applied Biosystems 3130 3130x and 3500 3500xL instruments Yfiler Plus PCR Amplification Kit User Guide 57 Analyze Data Set up GeneMapper ID X Software for data analysis If you need to create your own size standard definition use the following procedure to create the size standard definition file 1 Select Tools GeneMapper ID X Manager to open the GeneMapper ID X Manager 2 Select the Size Standards tab then click New GeneMapper ID X Manager
125. rom the heat block or oven 5 Let the lysate stand at room temperature for at least 15 minutes to cool the lysate for accurate pipetting 6 Transfer the sample lysate out of the 1 5 mL tubes or sample plate into tubes or plates for storage then discard the 1 5 mL tubes or deep well plate containing the swab heads Note To minimize the risk of contamination do not remove the swab heads from the sample lysate plate before transferring the lysate 7 Proceed to the next section to prepare the reactions or see Store the sample lysate on page 31 Prepare the 1 Add Prep n Go Buffer Part no 4471406 to the control wells in the reaction reactions plate Well s Add the following to wells of a MicroAmp Optical 96 Well Reaction Plate Negative control 3 uL of Prep n Go Buffer Positive control e For 25 and 26 cycles 0 uL of Prep n Go Buffer e For 27 cycles 1 uL of Prep n Go Buffer For 28 cycles 2 uL of Prep n Go Buffer 2 Prepare reagents a Thaw the Master Mix and Primer Set then vortex for 3 seconds and centrifuge briefly before opening the tubes or bottles IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore do not require subsequent thawing Do not refreeze the reagents Yfiler Plus PCR Amplification Kit User Guide 29 Swab substrates prepare reactions 30 3 Calculate the volume o
126. s user supplied 20 references 119 run module electrophoresis 35 41 S safety biohazard 116 chemical 116 Safety Data Sheets SDSs obtaining 123 sample discs size 26 sample preparation 23 DNA negative control 23 DNA positive control 23 standards 17 software instrument compatibility 15 spectral calibration 6 dye 38 43 stutter check version 47 import 48 stutter filters plus and minus 69 stutter peaks plus and minus 69 support obtaining 124 swab substrates prepare for PCR 28 Yfiler Plus PCR Amplification Kit User Guide Index T technical support 124 thermal cyclers for use with kit 114 programming conditions 23 31 training information on 124 treated paper 26 treated paper prepare for PCR 26 U untreated paper prepare for PCR 26 user supplied reagents 20 V validation characterization of loci 81 developmental 63 experiments to evaluate 63 importance of 63 mixture studies 87 sensitivity 83 thermal cycler parameters 65 W warranty 124 Whatman FTA Cards 9 work area amplified DNA 114 PCR setup 113 setup and lab design 113 workflow overview 13 14 127 Index 128 Yfiler Plus PCR Amplification Kit User Guide Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support technologies www lifetechnologies com 30 October 2014
127. s that do not size within the prescribed window around an allelic ladder allele It is important to note that while the precision within a set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of parameters including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also be found between runs on the same instrument and between runs on different instruments because of these parameters We strongly recommend that the allele sizes obtained be compared to the sizes obtained for known alleles in the Yfiler Plus Allelic Ladder from the same run and then converted to genotypes For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 Extra Peaks in the electropherogram Causes of extra peaks Stutter products Peaks other than the target alleles may be detected on the electropherogram displays Several causes for the appearance of extra peaks including the stutter product at the n 4 position incomplete 3 A nucleotide addition at the n 1 position artifacts and mixed DNA samples see SWGDAM Guideline 3 8 on page 87 Stutter is a well characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller or less frequently one repeat larger than the major STR product Butler 2005 Mulero et al 2006 S
128. shold Peak Start 0 0 a Peak End 0 0 5 2nd Order Least Squares L gt 3rd Order Least Squares Normalization D Cubic Spline Interpolation E Use Normalization if applicable Local Southern Method 5 Global Southern Method Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of Yfiler Plus Kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Size calling method The Yfiler Plus Kit has been validated using the Local Southern sizing method 60 460 base pairs Select alternative sizing methods only after performing the appropriate internal validation studies e Normalization A Normalization checkbox is available on this tab in GeneMapper ID X Software for use in conjunction with data run on the 3500 Series Genetic Analyzers Yfiler Plus PCR Amplification Kit User Guide 55 Analyze Data Set up GeneMapper ID X Software for data analysis Peak Quality tab settings 56 Analysis Method Editor General Allele Peak Detector Homozygo
129. st results Accuracy is the degree of conformity of a measured quantity to its actual true value Accuracy of a measuring instrument is the ability of a measuring instrument to give responses close to a true value SWGDAM December 2012 Laser induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2000 and Wallin et al 2002 However accuracy and reproducibility of Yfiler Plus Kit profiles have been determined from various sample types Figure 10 shows the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3500xL Genetic Analyzer with POP 4 polymer The X axis represents the nominal nucleotide sizes for the Yfiler Plus Allelic Ladder The dashed lines parallel to the X axis represent the 0 5 nt windows The Y axis represents the deviation of each sample allele size from the corresponding Yfiler Plus Allelic Ladder allele size All sample alleles are within 0 5 nt from a corresponding allele in the Yfiler Plus Allelic Ladder Yfiler Plus PCR Amplification Kit User Guide 67 5 Experiments and Results Accuracy precision and reproducibility Precision and size windows 68 Figure 10 Size deviation of 78 samples analyzed on the 3500xL Genetic Analyzer Precision 3500xl Marker DYF387S1 DYS19 DYS385 DYS389I DYS389II DYS390 DYS391 DYS392 DYS393 DYS437 DYS438 DYS439 DYS4
130. t and DYS481 right loci 200 220 205 225 245 soo 000 zooo 7000 sooo 000 sooo sooo 3000 3000 2000 2000 eo 4000 Yfiler Plus PCR Amplification Kit User Guide 77 5 Experiments and Results Extra Peaks in the electropherogram Addition of 3 A 78 Many DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This non template addition results in a PCR product that is one base pair longer than the actual target sequence and the PCR product with the extra nucleotide is referred to as the A form Figure 19 The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Yfiler Plus Kit includes two main design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 22 minutes This final extension step gives the DNA polymerase additional time to complete A addition to all double stranded PCR products See Figure 19 for an example of incomplete and normal A addition Final extension incubation for longer than the recommended 22 minutes may result in double A addition in which two non template adenosine residues are added to the PCR product Double A addition can cause shoulders on the
131. t occurs only once in a given population The number of unique haplotypes is usually less than the number of different haplotypes in any given population Table 7 Discriminatory capacity and number of unique haplotypes for four U S populations African American U S Caucasian U S Hispanics U S Asian Y STR marker N 557 N 533 N 391 N 340 combination DC UH DC UH DC UH DC UH Yfiler 98 2 547 95 7 510 95 9 375 91 5 311 Yfiler Plus 99 6 555 98 5 525 98 0 383 94 4 321 Mutation rate The most accurate method of estimating Y STR mutation rates is the direct observation of transmission between father and son A large scale Y STR analysis of mutation rates was performed with 2000 DNA confirmed father son pairs and encompassed the Yfiler Plus marker set Ballantyne et al 2010 2012 and 2014 Yfiler Plus PCR Amplification Kit User Guide 91 5 Experiments and Results Mutation rate 92 Yfiler Plus PCR Amplification Kit User Guide A Table of typical precision results Table of Precision Results Table 8 Example of precision results of seven injections of the Yfiler Plus Allelic Ladder run on the 3130x 3500 and 3500xL Genetic Analyzers 3130xl 3500 3500xL Allele Standard Standard Standard Mean deviation Mean deviation Mean deviation DYF387S1 30 264 16 264
132. tain and run the HID Updater eee 36 Create a Yfiler Plus assay 2 0 0 kk kk kk kk kk kk kk kK KK KK kk kK KK kK kk kk kk kk kk kk kk kk k 36 Modify 3500 QC protocol size calling method 0 kk KK KK KK KK KK KK KK KK KK KK 36 Perform spectral calibration kk kk kk kk kk kK kk KK kK KK KK KK KK KK KK KK KK KK KK KK kk k 38 Prepare samples for electrophoresis on the 3500 3500xL instruments nn 38 Section 3 2 3130 3130xl instruments attente tan KK KK KK KK KK kk 41 Set up the 3130 3130xl instruments for electrophoresis oen KK KK eee 41 Reagents and parts Jk kk kk kk kk kk kK kk kk kk kK kK kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk k 41 Electrophoresis software setup and reference documents ooo 41 Obtain and activate the 6 dye license for the instrument neen 41 Perform spectral calibration kk kk kk kk kK kK kk KK KK KK KK KK KK KK KK KK KK KK KK kK kk k 43 Prepare samples for electrophoresis on the 3130 3130xl instruments nnn 43 CHAPTER 4 Analyze Data 0 cece KK IK KK kk kk kk kk ok 45 Overview of GeneMapper D X Software V1 4 neen 45 oud a SEN N leeren dare teed Sealer eens ed ae Ae ed E 45 Before you Start reniei eal reni near en ie EEE UREN EE AE 45 Set up GeneMapper D X Software for data analysis kk KK KK KK een 47 Panel bin and stutter file version eenen eeen 47 Before using the software for the first time nennen 47 Check panel bin and stutter fil
133. tation and Support Related documentation Document title in Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide 4352716 Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Applied Biosystems 3500 3500xL Genetic Analyzer Quick Reference Card 4401662 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Data Collection v2 0 4476988 Applied Biosystems 3500 3500xL Genetic Analyzer User Bulletin Solutions to issues related to software data 4445098 hardware and consumables Note Additional user bulletins may be available at www lifetechnologies com GeneAmp PCR System 9700 Base Module User s Manual N805 0200 Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation User Bulletin 4440754 Quantifiler HP and Trio DNA Quantification Kits User Guide 4485354 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit User s Manual PrepFiler Forensic DNA Extraction Kit User Guide 4390932 GeneMapper ID X Software Version 1 2 Reference Guide 4426481 GeneMapper ID X Software Version 1 2 Quick Referenc
134. ter installs plate templates assays and instrument protocols that can be used to run Yfiler Plus Kit samples For more information refer to the release notes provided with the Updater Note If you have a new instrument installed by a Life Technologies representative the updater may have been run during installation 1 Shut down the 3500 3500xL Data Collection Software 2 Download the updater from www lifetechnologies com support gt Software Patches amp Updates gt 3500 Series Genetic Analyzers for Human Identification HID Updater 3500 DC v2 0 Click on the Read me file to review the software release notes 3 4 Click on the updater exe file 5 Follow the on screen prompts 6 Restart the computer The Yfiler Plus assay will have an instrument protocol and a QC protocol The easiest way to create an assay is to start off with an existing one that can be modified independently To modify an existing assay 1 Access the Assays library by selecting the Library tab and then the Assays tab under Library Resources 2 Select an existing assay i e GF_POP4 click duplicate and give it anew name i e YFP_POP4 3 Select the new assay and then click Edit to open it 4 Open the instrument protocol by clicking on Edit and modify the injection and run conditions specific to your instrument class and as shown on the table on page 35 Save the modified instrument protocol by clicking on Save to Library and give
135. the capability of the kit reagents to generate a profile of expected genotype The DNA Control 007 is not designed to be used as a DNA quantitation control and you may see variation from the labelled concentration when quantitating aliquots of the DNA Control 007 Standards for samples For the Yfiler Plus Kit the panel of standards needed for PCR amplification PCR product sizing and genotyping are DNA Control 007 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the Yfiler Plus Allelic Ladder e GeneScan 600 LIZ Size Standard v2 0 Used for obtaining sizing results This standard which has been evaluated as an internal size standard yields precise sizing results for Yfiler Plus Kit PCR products Order the GeneScan 600 LIZ Size Standard v2 0 Part no 4408399 separately e Yfiler Plus Allelic Ladder Allelic ladder developed by Life Technologies for accurate characterization of the alleles amplified by the Yfiler Plus Kit The Yfiler Plus Allelic Ladder contains most of the alleles reported for the 27 loci Refer to Table 1 on page 10 for a list of the alleles included in the Yfiler Plus Allelic Ladder Yfiler Plus PCR Amplification Kit User Guide 17 1 Overview Materials and equipment 18 Yfiler Plus PCR Amplification Kit User Guide Perform PCR m Section 2 1 Amplification from extracted DNA oo 20 Required user supplied reagents oen
136. the protocol a new name 5 Follow the instructions below to edit the QC protocol The Yfiler Plus Kit has been validated with data that was analyzed using the Local Southern method 60 460 base pairs The QC protocol provided in the HID assay installed by the HID Updater 3500 DC v2 0 is set for the 3rd Order Least Squares method To use the Local Southern method for fragment sizing edit the QC protocol 1 In the Library tab open the QC Protocol window Yfiler Plus PCR Amplification Kit User Guide Section 3 1 3500 3500xL instruments 3 Set up the 3500 3500xL instruments for electrophoresis 2 Create a new QC protocol according to the figure Setup a QC Protocol Protocol Name lt Description Size Standard GS600 _LIZ_ 60 460 Sizecaller SizeCaller v1 1 0 Analysis Settings qc Settings Analysis Range Full z Sizing Range Partial zj lt Size Calling Method Local Southern xj Analysis Start Point 0 Sizing Start Size 60 lt Analysis Stop Point 1000000 Sizing Stop Size 460 lt 7j Green W Yellow V Red Peak Amplitude Threshold EN 175 175 175 Common Settings Use Smoothing Light v Use Baselining Baseline Window Pts V 33 Minimum Peak Half Width 2 Peak Window Size 13 Polynomial Degree 3 Slope Threshold Peak Start 00 Slope Threshold Peak End 00 a Name the new QC protocol according to your laboratory s standard convention b Set the following parameters
137. tial amplification was observed in the denaturation temperature experiments Of the tested annealing temperatures 61 61 5 and 62 C produced robust profiles with no significant cross reactivity to 1 ug of female DNA At 62 5 C the yield of the majority of loci was significantly reduced This should pose no problem with routine thermal cycler calibration and when following the recommended amplification protocol Preferential amplification was not observed at the standard annealing temperature of 61 5 C Figure 7 Amplification of a mixture of 1 ng of male 007 DNA and 1 ug of female 9947 DNA at annealing temperatures of 60 5 61 61 5 62 62 5 and 63 C analyzed on an Applied Biosystems 3500xL Genetic Analyzer Y axis scale is 0 to 8 000 RFU Mare Sampie for Deletion all a lil oe Meh deli akd Maric Sample for Deletion swo iie a ai al aad SaS 7 j 7 i 61 5 C idla ll Alde aili ud d 7 5 En T 62 C En ed eae a NIYEN 62 5 C idg w di ki Jl ili al J j j 63 C lee EN da td Aala dd d Figure 8 Electropherograms obtained from amplification of a blood sample on an FTA card at annealing temperatures of 60 5 61 61 5 62 and 62 5 C analyzed on an Applied Biosystems 3500xL Genetic Analyzer Y axis scale is 0 to 12 000 RFU E Mark Semple for Deletion T j j i i l 60 5 C _lli ri
138. ties of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 Also take care while handling and processing samples to prevent contamination by human DNA Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology PCR setup work area IMPORTANT These items should never leave the PCR Setup Work Area Calculator e Gloves disposable e Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack e Pipette tips sterile disposable hydrophobic filter plugged Pipettors Yfiler Plus PCR Amplification Kit User Guide 113 PCR Work Areas Amplified DNA work area Tube decapper autoclavable e Vortex Amplified DNA work area IMPORTANT Place the thermal cyclers in the Amplified DNA Work Area You can use the following systems GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block IMPORTANT The Yfiler Plus Kit is not validated for use with the GeneAmp PCR System 9700 with the Aluminium 96 Well Block Use of this thermal cycling platform m
139. tion Kit User Guide Chapter 4 Analyze Data 4 Set up GeneMapper ID X Software for data analysis General tab settin gs Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings Analysis Method Description Yfiler Plus Analysis Method GeneMapper ID X Security Group In the Name field either type the name as shown or enter a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the dropdown list The Description and Instrument fields are optional Yfiler Plus PCR Amplification Kit User Guide 53 Analyze Data Set up GeneMapper ID X Software for data analysis Allele tab settings 3 Analysis Method Editor General Allele peak Detector Peak Quality SQ amp GQ Settings Bin Set Yfler_Plus_Bins_v1 Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Global Cut off Value 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Global Minus Stutter Ratio 0 0 0 0 Global Minus Stutter Distance From 0 0 3 25 To 0 0 4 75 Global Plus Stutter Ratio 0 0 0 0 Global Plus Stutter Distance From 0 0 0 0 To 0 0 0 0 Amelogenin Cutoff 0 0 Range Filter Factory Defaults The settings shown in the screen shot above were used during the developmental validation of the Yfiler Pl
140. ture studies The ability to obtain reliable results from mixed source samples should be determined SWGDAM December 2012 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the results We recommend that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing system is able to detect DNA mixtures In the case of Y STRs the female DNA component is not amplified by the Y chromosome specific primers Male female mixture studies were performed up to a ratio of 1 4000 using three different female DNAs The amount of female DNA was kept constant at 1 ug and the amount of male control DNA was changed The female DNA did not cause any interference with the interpretation of the male Y STR profile as shown in Figure 25 Low level artifacts with female DNA have been occasionally observed in the FAM 270 280 bp and TAZ 225 260 bp dye In general these artifacts peaks should not affect interpretation due to their morphology and intensity Figure 25 Amplification of male Contro
141. ular loci result from differences in the number of 4 bp repeat units We have sequenced all the alleles in the Yfiler Plus Allelic Ladder In addition other groups in the scientific community have sequenced alleles at some of these loci Redd et al 2002 www cstl nist gov biotech strbase y_strs htm Among the various sources of sequence data on the Yfiler Plus Kit loci there is consensus on the repeat patterns and structure of the STRs Mulero et al 2014 Gusmao et al 2006 The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 39 families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992 Three CEPH family DNA sets were examined 1 ng of DNA from each sample was amplified using the Yfiler Plus Kit and the Identifiler Kit followed by analysis using a 3500xL Genetic analyzer The families examined included 1333 9 offspring 7 males 1340 7 offspring 5 males and 1345 7 offspring 5 males representing 23 meiotic divisions The Identifiler Kit results confirmed that the loci are inherited according to Mendelian rules as reported i
142. uorescent dye colors into distinct spectral components The five dyes used in the Yfiler Plus Kit to label samples are 6 FAM VIC NED TAZ and SID dyes The sixth dye LIZ dye is used to label the GeneScan 600 LIZ Size Standard v2 0 Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the Life Technologies instruments the fluorescence signals are separated by diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 6 FAM dye emits at the shortest wavelength and it is displayed as blue followed by the VIC dye green NED dye yellow TAZ dye red SID dye purple and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 3 The goal of multicomponent analysis is to correct for spectral overlap Yfiler Plus PCR Amplification Kit User Guide 15 1 Overview Instrument and software overview Figure 3 Spectral calibration of the six dyes used in the Yfiler Plus Kit LIz TAZ NED VIC 6 FAM SID 16 Yfiler Plus PCR Amplification Kit User Guide Chapter 1 Overview Materials and equipment Materials and equipment Kit contents and The Yfiler Plus PCR Amplification Kit is available in two sizes storage 100 reactions Cat n
143. urce Yfiler Plus PCR Amplification Kit 100 reaction 4484678 Yfiler Plus PCR Amplification Kit 500 reaction 4482730 Prep n Go Buffer 5 tubes 1000 tests 4467079 Prep n Go Buffer for buccal swabs 200 reactions 4471406 3130 Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130x Genetic Analyzer Capillary Array 36 cm 4315931 POP 4 Polymer for 3100 3100 Avant Genetic Analyzers 4316355 GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 402824 Hi Di Formamide 4311320 DS 36 Matrix Standard Kit Dye Set J6 4425042 MicroAmp Optical 96 Well Reaction Plate N8010560 3130xl Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130x Genetic Analyzer Capillary Array 36 cm 4315931 POP 4 Polymer for 3130 3130x Genetic Analyzers 4352755 GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10x 402824 DS 36 Matrix Standard Kit Dye Set J6 4425042 MicroAmp Optical 96 Well Reaction Plate N8010560 Hi Di Formamide 4311320 For a complete list of parts and accessories for the 3130xl instrument refer to Appendix A of the 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Pub no 4352716 3500 3500xL Analyzer materials Anode buffer container ABC 4393927 Cathode buffer container CBC 4408256 POP 4 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 POP 4 polymer 38
144. us Kit To specify settings e In the Bin Set field select the Yfiler Plus Bins _v1 bin set e GeneMapper ID X Software v1 0 1 or higher allows you to specify 4 types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column e Specify the appropriate filter settings To apply the stutter ratios contained in the Yfiler_Plus_Stutter_v1 file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Peak Detector tab IMPORTANT The Local Southern Sizing algorithm has been validated for analysis of settings Yfiler Plus Kit data on 3130 and 3500 series instruments 54 Yfiler Plus PCR Amplification Kit User Guide Chapter 4 Analyze Data 4 Set up GeneMapper ID X Software for data analysis Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings Peak Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Full Range AllSizes X B R Start Pt 0 Start Size 0 Stop Pt 10000 Stop Size 1000 G P ve 0 rSmoothing and Baselining Min Peak Half Width 2 pts Smoothing None m Light Polynomial Degree Heavy Peak Window Size 13 pts Baseline Window 33 pts Slope Thre
145. us min peak height Max Peak Height MPH Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs Allele Number AN Max expected alleles For Y markers Allelic Ladder Spike Spike Detection Cut off Value Sample Spike Detection Spike Detection Min Max Peak Height LPH MPH Heterozygous min peak height For autosomal markers amp AMEL Perform internal validation studies to determine settings IMPORTANT Perform the appropriate internal validation studies to determine the heterozygous and homozygous minimum peak height thresholds maximum peak height threshold and the minimum peak height ratio threshold for interpretation of Yfiler Plus Kit data Yfiler Plus PCR Amplification Kit User Guide SQ amp GQ tab settings Create a size standard Chapter 4 Analyze Data Set up GeneMapper ID X Software for data analysis Analysis Method Editor gt n General Allele Peak Detector Peak Quality SQ amp GQ Settings Quality weights are between 0 and 1 Sample and Control GQ Weighting Broad Peak BD 0 8 Allele Number AN 1 0 Out of Bin Allele BIN 0 8 Low Peak Height LPH 0 3 Overlap OVL 0 8 Max Peak Height MPH 0 3 Marker Spike SPK 0 3 Off scale OS 0 8 AMEL Cross Check ACC 0 0 Peak Height Ratio PHR 0 3 Control Concordance CC Weight 1 0 Only applicable to controls SQ Weighting B
146. using the Yfiler Plus Kit for the first time perform a single initial sensitivity experiment to determine the appropriate cycle number to use during internal validation studies and operational use of the Yfiler Plus Kit This experiment accounts for instrument to instrument and sample to sample variations If you are processing multiple sample type and substrate combinations for example buccal samples on treated paper and blood samples on untreated paper perform separate sensitivity experiments for each sample type and substrate to be used for testing The Yfiler Plus Kit is optimized to amplify unpurified e Single source blood samples on treated paper e Buccal samples on treated paper substrates without the need for sample purification e Blood samples collected on untreated paper with the addition of Prep n Go Buffer e Buccal samples collected on swab substrates and treated with Prep n Go Buffer When amplifying single source unpurified samples using the Yfiler Plus Kit you should expect to see greater variation in peak height from sample to sample than is expected with purified samples Careful optimization of the cycle number will help to minimize the impact of this variation 1 Select 20 of each sample and substrate type Ensure the selected samples represent a typical range of samples analyzed in your laboratory IMPORTANT The number of samples recommended for this study has been chosen to allow you to c
147. w assuming the analysis is complete Yfiler Plus PCR Amplification Kit User Guide Chapter 4 Analyze Data 4 For more information For more information For more information refer to GeneMapper ID X Software v1 4 New Features and Installation Procedures User Bulletin Pub no 4477684 GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide Pub no 4396773 GeneMapper ID X Software Version 1 2 Reference Guide Pub no 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide Pub no 4426482 Yfiler Plus PCR Amplification Kit User Guide 61 JA Analyze Data For more information 62 Yfiler Plus PCR Amplification Kit User Guide Overview Importance of validation Experiment conditions Experiments and Results OVERVIEW rijen Sunda HHHH Bb Ey MMMIJ M O 63 Developmental validation LA AKS KK KK KK KK KK KK KK KK KK KK KK KK KK K 64 Accuracy precision and reproducibility SK KK KK KK KK 67 Extra Peaks in the electropherogram ooo 69 Characterization of loci eee KK KK KK eee eee 81 Specles spEilENEY set on ate stenen eten dar K dy kin Lane et ale 82 SENSI senen sees he ase ae aten ed Parted 83 Stability ws pases bb a
148. x into each reaction well of a MicroAmp Optical 96 Well Reaction Plate Yfiler Plus PCR Amplification Kit User Guide 27 Swab substrates prepare reactions 7 Swab substrates Sample prep guidelines Prepare the 1 sample lysate room temperature 9 protocol Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells place a MicroAmp compression pad Cat no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders prepare reactions Detach each buccal swab head from the swab shaft before lysis If using the heated lysis protocol perform lysis in either of the following formats 1 5 mL tubes with a heat block VWR Scientific Select dry heat block or similar 96 well deep well plate Part no 4392904 with an oven and a metal plate adaptor Robbins Scientific Model 400 Hybridization Incubator or similar Agilent Benchtop Rack for 200 ul Tubes V Bottom Plates metal Part no 410094 or similar IMPORTANT Do not use a plastic plate adaptor For optimum performance lysis of a whole swab is recommended To preserve the sample evaluate lysis of
149. y Life Technologies and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 124 Yfiler Plus PCR Amplification Kit User Guide Numerics 3130 3130xl instruments 41 3500 3500xL instruments 34 6 dye license activation 41 spectral calibration 38 43 A A nucleotide addition by AmpliTaq Gold to 3 end of amplicon 78 agarose gel using to examine DNA 85 allele frequencies in the population databases 89 allelic ladder about 17 profile 11 requirements for accurate genotyping 33 volume per reaction 38 44 amplification differential amplification of loci 85 loci 10 artifacts 79 B bins check version 47 import 48 biohazard safety 116 blood samples 25 26 Bode Buccal DNA Collector 9 buccal samples 25 26 buccal swabs 28 c chemical safety 116 contents of kit 17 control DNA 17 control DNA 007 12 17 Yfiler Plus PCR Amplification Kit User Guide Index D Data Collection Software overview 15 data analysis 90 degraded DNA 85 developmental validation 63 differential amplification of loci 85 discriminatory capacity 91 DNA control about 17 effect of DNA quantity on results 83 how degraded DNA affects which loci amplify 85 negative control sample preparation 27 29 ne
150. y is improved over Quantifiler Duo Human and Y Human Male DNA Quantification Kit assays The primary quantification targets Small Autosomal and Y consist of relatively short amplicons 75 to 80 bases to improve the detection of degraded DNA samples In addition kits each contain a Large Autosomal target with a longer amplicon gt 200 bases to aid in determining if a DNA sample is degraded Prepare the amplification kit reactions 22 Calculate the volume of each component needed to prepare the reactions using the table below DNA sample Volume per reaction Yfiler Plus Master Mix 10 0 uL Yfiler Plus Primer Set 5 0 HL Prepare reagents Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers Thaw the Master Mix and the Primer Set then vortex all reagent tubes including the enzyme for 3 seconds and centrifuge briefly before opening the tubes IMPORTANT Thawing is required only during first use of the Primer Set and Master Mix After first use these reagents are stored at 2 to 8 C and therefore they do not require subsequent thawing Do not refreeze these reagents Prepare the reaction mixture Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the reaction mixture for 3 seconds then centrifuge briefly Dispense 15 uL of the reaction mixture into each reaction well of a
151. ycles 1 uL of Control DNA 007 Note The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number The final volume in each well is 28 uL reaction mix plus Prep n Go Buffer and sample lysate or positive control Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film IMPORTANT We recommend adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation Do not use caps which may not provide a consistent seal across all wells Yfiler Plus PCR Amplification Kit User Guide Section 2 2 Direct amplification of DNA 2 Perform PCR IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block place a MicroAmp compression pad Part no 4312639 on top of the plate to additionally prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad 9 Vortex the reaction mix at medium speed for 3 seconds 10 Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders 11 Amplify the samples in a Veriti 96 well Thermal Cycler or PCR System 9700 with the silver or gold plated silver 96 well block as described in Perform PCR on page 31 Store the sample Cap the sample lysate storage tubes or seal the sample lysate storage plate with lysate Mic

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Yfiler&reg; Plus PCR Amplification Kit User Guide

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Yfiler® Plus PCR Amplification Kit User Guide