Comparison of the Promega Power Y and AB AmpFlSTR YFiler kits and the Subsequent Validation of YFiler.

Transcription

1 Comparison of the Promega Power Y and AB AmpFlSTR YFiler kits and the Subsequent Validation of YFiler. Dr. Timothy P McMahon Armed Forces DNA Identification Laboratory Rockville, Md Timothy.mcmahon@afip.osd.mil

2 European Minimal haplotype SWGDAM Additional Loci

3 Allelic Ladder

4 Amplification Conditions Water Gold Star 10x Buffer 10X Primer pair Mix TaqGold (5units/ul) DNA Total reaction volume 9.45 to 18.45ul 2.5ul 2.5ul 0.55ul 1 to 10ul 25ul Amplification of >2ng leads to locus to locus imbalance Store templates in TE-4 If DNA stored in TE should not exceed 20% final volume 32 cycles works for 0.5 to 1ng of DNA Use a Female control negative and a PCR Negative

5 Promega Notes Selected primers to minimize stutter Modified primer sequences and added a final extension to prevent minus A peaks Amplify 0.5 to 1ng of input DNA, more template stochastically amplify smaller loci over larger loci. Follow suggested parameters

6 PowerPlex Y Allelic Ladder DYS391 DYS389I DYS439 DYS389II DYS438 DYS437 DYS19 DYS392 DYS393 DYS390 DYS385

7 YFiler Kit Specific Information

8 Power of Discrimination

9 Amplification Conditions AmpFlSTR Y-Filer PCR Reaction Mix 9.2μl AmpFlSTR Y-Filer Primer set 5.0μl AmpliTaq Gold Polymerase 0.8μl Template DNA 1-10μl DH2O q.s. 25μl Hold 95 C for 11 minutes, then: 30 Cycles 94 C for 1 minute, 61 C for 1 minute 72 C for 1 minute Samples resuspended in TLE Hold Hold 60 C for 80 minutes 4 C soak 1ng of input DNA optimal

10 Y-Filer Allelic Ladder DYS456 DYS389I DYS390 DYS389II DYS458 DYS19 DYS385 DYS393 DYS391 DYS439 DYS635 DYS392 H4 DYS437 DYS438 DYS448

11 Comparison Experiment Dilutions were made Quantified Amplified using PowerPlex Y or AmpFlSTR- YFiler at 30 and 32 cycles Analyzed on the AB-3100 Genetic Analyzer

12 30_cycle_5sec_1ng DYS391 DYS385

13 30_cycle_5sec_100pg

14 30_cycle_5sec_50pg

15 32_cycle_5sec_1ng

16 Validation of AmpFlSTR Y-FilerY

17 Validation Outline Interplate precision Sensitivity and Reproducibility Mixtures Stutter Non-Probative Casework Samples Analysis on the AB-3100 Genetic Analyzer

18 Precision Allelic Ladders were injected for 2, 5, and 10 seconds and analyzed on a 36 cm Array using POP4. 39 Ladders were analyzed at the 2, 5 and 10 second injections The base-pair size for each allele was determined and a final table was exported. % precision was calculated by dividing the std dev by the mean base pair size

19 2 second injection 5 second injection 10 second injection

20 DYS385

21 Conclusion The Average Standard Deviations for all loci ranged between and 0.085, which was consistent with the precision results published in the Y-Filer Y user manual. The Y-Filer Y ladder was greater than 99.9% at a 5 second injection for all 17 loci The 2 and 10 second injections had greater than 99.9% for all loci except DYS456 which was 99.88% Results support the use of a 5 second injection as the default injection.

22 Sensitivity 9948 was serially diluted. Quantitated and normalized Amplified in duplicate by two scientist (4 replicates. Samples injected at 2, 5 and 10 seconds Analyzed at a GeneScan setting of 35.

23

24 Sensitivity results expressed as relative fluorescent units AB determined the Average peak height for 3 replicates of a 1ng sample was AFDIL s average peak height for four 1ng replicates was 1613 This was less than a 1.2 fold difference between results

25 DYS439 DYS456

26 Increasing Injection Time

27 Conclusions Targeted input of 1ng in 1 to 10 l l of DNA 5 second injection default Minimum Reporting Threshold of 100 RFU Maximum RFU of 6000 Linear increase in RFUs with increasing injection times Results consistent with published results

28 Stutter

29 Mixtures Full profiles above 0.25ng and partial profiles below. No non-specific amplification products detected Peak heights consistent with peak heights observed during sensitivity 100ng of Female DNA competitive inhibition observed at random loci No non-specific peaks detected

30 Male:Male Mixtures 1:4

31 Reproducibility using Non-Probative Case Samples 0.5 or 1.0 ng of input DNA

32 Tissue Samples 0.65ng/ l ~1.4 fold difference in RFUs is within the realm of typing plate set up and injection Variations. The 1.8 and 2.0 fold difference was due to the analysts using different template volumes

33 Organic Bone 04T A1-1_500 04T A1-1_500

34 Bloodstains

35 Paternity

36 Conclusions AmpFlSTR STR-Y-Filer kit was proven to be acceptable for use at the AFDIL\ Kit was sensitive from 0.25 to 5ng Detect mixture from a 19:1 to a 4:1 ratio Precision was greater than 99.9% at 5 seconds Little to no variation was observed during the reproducibility and non-probative portion. Results were concordant with Reliagene Y 6 results and between different individuals Y-Filer kit generated conclusions that were consistent with previous conclusions i.e. exclusions vs. inclusions Results concordant with AB published results

37 Acknowledgements Robert Oliver Dr. Pattie Foley Erica Chatfield Faith Patterson Robin Yaghmour Demris Lee

38 Placement of this presentation on Applied Biosystems Website does not constitute an endorsement of the company by the Armed Forces DNA Identification Laboratory or the Department of Defense.