Integrating the Quantifiler Duo DNA. Yfiler PCR Amplification Kit in Sexual Assault Investigations

Transcription

1 Integrating the Quantifiler Duo DNA Quantification Kit with the AmpFlSTR Yfiler PCR Amplification Kit in Sexual Assault Investigations Lisa Calandro, M.P.H. Sr. Manager, Technical Marketing

2 Outline Introduction Y STR analysis Quantifiler Duo assay specificity and efficiency Performance with mixture samples Performance with inhibited and degraded samples Correlation to AmpFlSTR Yfiler kit performance Conclusions Applied Biosystems

3 Y STR Analysis

4 When Are Y STRs Used in Forensic Casework?* Autosomal testing yields little or no male DNA profile Samples where female DNA is in vast excess of male DNA Low level minor male - interpretation tricky or impossible Very few or no sperm are detected Differential extraction is not possible non sexual assault samples Clarify the number of male contributors Last resort type of testing: Cold cases Post-conviction cases *Source: Cassie Johnson, Orchid Applied Biosystems

5 Detecting Male DNA in Forensic Samples Autosomal analysis may reveal presence of small amelogenin Y peak However, autosomal analysis does not always reveal presence of male DNA in mixed samples Quantifiler Y quantitation has capability to specifically detect male DNA in the presence of >1:9000 female:male mixture ratio Applied Biosystems

6 AmpFlSTR Yfiler Amplification Kit Overview Amplifies 17 loci in a single reaction using 5-dye chemistry for increased discrimination capacity Same dye set used with the AmpFlSTR Identifiler Kit Provides specific and sensitive detection of male DNA in the presence of high concentrations of female DNA Developed and validated specifically with the Quantifiler Human and Y Human Male Assays User-friendly Yfiler TM haplotype database Validated for use with ABI PRISM 310, 3100, 3100-Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Applied Biosystems

7 AmpF STR Yfiler Kit 17 Y-STR Loci Applied Biosystems Y DYS19 DYS389I/II DYS390 European DYS391 Minimal DYS392 Haplotype DYS393 DYS385a/b DYS437 DYS438 SWGDAM DYS439 DYS448 DYS456 ABI DYS458 DYS635 Y GATA H4

8 Identifiler Analysis of Casework Sampleª * Lip/breast swabbing from female victim of oral assault * Since no male DNA detected, a range of input DNA will ªSource: be Joe amplified Varlaro, Spencer in YfilerLaboratories, Inc Applied Biosystems

9 Yfiler Analysis of Casework Sampleª Applied Biosystems

10 Why Y-STR Analysis? simulated forensic mixture sample 3 male DNA samples mixed at equal ratios Female DNA mixed with 3-male mixture at ratio of 500:1 Mixture sample quantified with both Quantifiler Human and Male assays 1ng input DNA added to an Identifiler reaction based on the Quantifiler Human results 1ng input DNA added to a Yfiler reaction based on the Quantifiler Male results Applied Biosystems

11 AmpFlSTR Identifiler Amplification Result sample quantified with the human assay only Amelogenin X only Only single source female sample detected Applied Biosystems

12 AmpFlSTR Yfiler Amplification Result sample quantified with the male assay Three male profiles detected Applied Biosystems

13 Discriminatory capacity for three U.S. populations Y-STR marker combination African American (N=786) Caucasians (N=778) Hispanics (N=381) European minimal haplotype (9) 75.8% 61.7% 79.8% US Haplotype (11) 86.8% 74.3% 85.6% US Haplotype + DYS437 (12) AmpF STR Yfiler kit (17) 87.6% 76.7% 88.2% 97.6% 95.5% 95.8% DC= (# of different haplotypes / pop. size) x Applied Biosystems

14 Most common Haplotype identified by the European Minimal Haplotype markers (20 individuals) Sample Info DYS19 1DYS385 DYS389 DYS389 DYS390 DYS391 DYS392 DYS393 DYS438 DYS439 DYS437 DYS448 DYS456 DYS458 Y GATA DYS635 C , C , ATCC 14 11, C , C , C , C , C , C276RL 14 11, C , C , C85RL 14 11, C , C , C , >13 24 C , C , C66RL 14 11, C , C , European Minimal Haplotype # of different 0 haplotypes SWGDAM Applied Biosystems Yfiler Kit 20

15 Samples from: Austria CA DOJ Italy Minn. BCA NIST OCME Orchid Poland Santa Clara County SeraCare SLED South Africa Yfiler Population Data by Ethnic Group Filipino 3% Hispanic 17% 3,561 Samples Asian 9% Caucasian 35% African American 28% Native American 3% Vietnamese 3% Black African 2% Applied Biosystems

16 Key Features of Yfiler Haplotype Database Ability to search all 17 Yfiler loci Ability to search microvariant Y STR types Ability to perform batch searches using outputs from either Genotyper or GeneMapper ID v Applied Biosystems

17 Microvariant Example Applied Biosystems Concordant with NIST 20plex and 11plex data

18 AmpF STR Yfiler Kit Input Haplotypes from Genotyper Search results for individual samples Applied Biosystems

19 Quantifiler Duo DNA Quantification Kit: Quantitative and Qualitative Assessment of Total Human and Human Male DNA

20 Objectives Develop a multiplexed real-time PCR assay to: Quantify total human DNA and human male DNA in a single PCR reaction Determine the ratio of human male and female DNA Detect PCR inhibitors Allow selection of appropriate STR amplification kit Predict success with downstream STR amplification Applied Biosystems

21 Quantifiler Duo Kit: A Guide to Sample Assessment DNA Extract IPC Ct Value Quantification Re-Purification Heavy Inhibition STR Analysis Mixture Ratio Autosomal STR Y-Filer kit MiniFiler kit Identifiler kit Partial Inhibition Applied Biosystems

22 Comparison to Quantifiler Kits DNA standard is pooled human male genomic DNA PCR Reaction Mix optimized for expanded multiplex performance Ability to obtain ratio of human and male DNA in a mixture sample in a single reaction Dual quantification of human and male DNA minimizes sample consumption Assay designed to guide selection of appropriate AmpFlSTR Kit chemistry Maximizes recovery of interpretable STR profiles Applied Biosystems

23 Configuration of Assay Target Marker Size Dye Human DNA Human Male DNA RPPH1 (Ribonuclease P RNA component H1) SRY (Sex determining region Y) 140 bp VIC 130 bp FAM IPC Artificial Template 130 bp NED The multiplex assay is run on the 7500 Real Time PCR System using SDS v1.2.3 software Applied Biosystems

24 Std. Curve Amplification Plots and ΔRn (SRY/RNaseP/IPC) SRY RPPH1 IPC Sample ng/µl Ave Ct Ave Ct Ave Ct Ave Rn Ave Rn Ave Rn RPPH1 SRY IPC RPPH1 SRY IPC std std std std std std std std Applied Biosystems

25 Standard Curves and IPC Ct Values Ave Ct Multiplex Standard Curve Human Genomic DNA SRY-FAM 24.0 RPPH1-VIC 22.0 IPC-NED Log of [ ] Applied Biosystems

26 RPPH1 Assay Specificity RPPH1 Male & Female Gorilla 10 ng of DNA from the indicated species was tested Applied Biosystems RNAse RPPH1P.6.4s (140bp) Species Ave.Ct Orangutan 40.0 Chimp 40.0 Gorilla 37.9 Macaque 40.0 Dog 40.0 Cow 40.0 Pig 40.0 Cat 40.0 Chicken 40.0 Fish (Salmon) 40.0 Rabbit 40.0 Mouse 40.0 Rat 40.0 Hamster 40.0 Male 26.5 Female 26.2 E.coli 40.0 Pseudomonas 40.0 Neisseria 40.0 Staphylococcus 40.0 Saccharomyces 40.0

27 SRY Assay Specificity SRY Male Chimp 10 ng of DNA from the indicated species was tested Applied Biosystems. SRY.2s 130 bp Species Ave. Ct Orangutan 40.0 Chimp 30.7 Gorilla 40.0 Macaque 40.0 Dog 40.0 Cow 40.0 Pig 40.0 Cat 40.0 Chicken 40.0 Fish 40.0 Rabbit 40.0 Mouse 40.0 Rat 40.0 Hamster 40.0 Male (Raji) 28.8 Female (NA14672a) 40.0 E.coli 40.0 pseudomonas 40.0 Neisseria 40.0 Staphylococcus 40.0 Saccharomyces 40.0

28 Analysis of Male & Female Mixtures Quantitation of Mixtures Quantity (ng/ul) SRY RPPH :0 1:2 1:5 1:10 1:20 Male/female DNA ratio Male DNA added at a constant concentration of 0.2 ng/µl Applied Biosystems

29 Mixture study: Identifiler kit profile (1 ng DNA/rxn) 1:0 Male component 1:2 1:5 1:10 1: Applied Biosystems 3000 rfu

30 Analysis of Male & Female Mixtures 50 pg of male DNA Male/female ratio SRY measured conc (ng/µl) RPPH1 measured conc (ng/µl) SRY/RPPH1 expected ratio SRY/RPPH1 measured ratio 1: :1 1:1 1: :2 1:2.4 1: :6 1:5.4 1: :11 1:10.6 1: :101 1:90 1: :501 1:408 1: :1001 1:1007 Male DNA added at a constant concentration of ng/µl Applied Biosystems

31 Mixture study: YFiler kit profile (1 ng DNA/rxn) M:F Ratio 1: rfu scale 1:100 1:500 1: Applied Biosystems

32 Post-coital samples Quantitation results (ng/μl) after differential extraction Quantitation of post-coital samples (e-cell fraction) Quantitation of post-coital samples (sperm fraction) Quantity (ng/ul) RPPH1 SRY Quantity (ng/ul) RPPH1 SRY E 2E 3E 4E S 2S 3S 4S Sample type Sample name RPPH1 SRY Quantifiler H Quantifiler Y E-cell fraction, 4h 1E E-cell fraction, 8h 2E E-cell fraction, 12h 3E E-cell fraction, 24h 4E Sperm fraction, 4h 1S Sperm fraction, 8h 2S Sperm fraction, 12h 3S Sperm fraction, 24h 4S Applied Biosystems

33 Post-coital samples: E cell - Identifiler kit profile (1 ng DNA/rxn) 2000 rfu 1E 4 h 2E 8 h 3E 12 h 4E 24 h Applied Biosystems

34 Post-coital samples: Sperm - Identifiler kit profile (1 ng DNA/rxn) 2000 rfu 1 S 4h 2 S 8h 3 S 12h 4 S 24h Applied Biosystems

35 Post-coital samples: sperm fraction - YFiler kit profile (1 ng DNA/rxn) 1 S 4h 2 S 8h 3 S 12h 4 S 24h Applied Biosystems 6000 rfu

36 Body fluid mixture samples - Quantitation results (ng/μl) Quantitation of body fluid mixture samples RPPH1 SRY Quantity (ng/ul) M 2M 3M 4M 5M 6M 7M Applied Biosystems SRY/RPPH1 measured ratio SRY/TERT measured ratio Sample type Sample name RPPH1 SRY Quantifiler Hu Quantifiler Y Male saliva ctrl 1M :1.1 1:0.87 Female saliva ctrl 2M Female blood ctrl 3M Male saliva/female saliva mix 1 4M :2.1 1:2.1 Male saliva/female saliva mix 2 5M :3.4 1:3.2 Male saliva/female blood mix 1 6M :3.2 1:3.7 Male saliva/female blood mix 2 7M :4.8 1:5

37 Mixture study conclusions Successful quantification of the male component in mixtures with male/female DNA ratios up to 1:1000 Tested using 25 pg/ul and 200 pg/ul of male DNA. Accurate measurement of the male/female ratio in a mixture sample relative to expected results DNA input based on Quantifiler Duo kit results led to generation of an interpretable profile of the male component up to a 1:10 male/female mixture ratio using the Identifiler kit DNA input based on Quantifiler Duo kit results led to generation of an interpretable YFiler kit profile for the male component up to a 1:1000 male/female mixture ratio Applied Biosystems

38 Effect of Inhibitors Evaluated the ability to detect the presence of known PCR inhibitors such as hematin and humic acid using the Quantifiler Duo kit Varied inhibitor concentrations in DNA samples and tested using the Quantifiler Duo and Identifiler kits to assess predictive performance Applied Biosystems

39 Humic Acid Inhibited Samples - IPC Signal Applied Biosystems

40 Humic Acid Inhibited Samples - SRY Signal 7.5ng/µl is undetected Applied Biosystems

41 Humic Acid Inhibited Samples - RPPH1 Signal 7.5ng/µl is undetected Applied Biosystems

42 Ctr-Hum_0 Humic Acid Inhibited Samples: Correlation with Identifiler Kit Profile Hum_1 ng/ul Hum_2 ng/ul IPC signal with increasing humic acid concentration Hum_3 ng/ul IPC Ct Hum_3.75 ng/ul Hum_7.5 ng/ul Hum_11.2 ng/ul 0ng/ul 1ng/ul 2ng/ul 3ng/ul 3.75ng/ul 7.5ng/ul ng/ul Concentration of humic acid 15ng/ul 30ng/ul Hum_15 ng/ul Applied Biosystems Hum_30 ng/ul

43 Ctr-Hem0 Hem_2.5 um Hematin Inhibited Samples: Correlation between IPC Signal and Identifiler Kit Profile Hem_5.0 um Hem_7.5 um IPC signal with increasing hematin concentration IPC Ct uM 2.5uM 5uM 7.5uM 10uM 12.5uM 15uM 17.5uM 20uM 40uM Concentration of Hematin Applied Biosystems Hem_10 um Hem_12.5 um Hem_15 um Hem_17.5 um Hem_20 um Hem_40 um

44 Inhibited Samples Quantifiler Duo kit results demonstrated IPC shift in the presence of increasing amounts of inhibitor Quantifiler Duo kit results correlated with STR kit performance Applied Biosystems

45 Degraded DNA Degraded DNA: Concentration (ng/ul) Concentration RNase RPPH1P 140. SRY 0.00 Intact 2 U 3U 4 U 5 U 6 U DNaseI Units Units Applied Biosystems

46 Quantification of Degraded DNA Results with Degraded DNA Series Quant Results (ng/ul) Quantifiler Human Quantifiler Y Multiplex RPPH1 Multiplex SRY 0.00 Intact 2u 3u 4u 5u 6u DNA Degradation State Quantification of degraded DNA was performed using Quantifiler Duo (Multiplex assay) and Quantifiler kits Applied Biosystems

47 Degraded DNA: Correlation with Identifiler Kit RPPH1 SRY Sample Human (ng/µl) Vol (µl) to give 1 ng STR Peaks Detected (out of 28) Male (ng/µl) Vol (µl) to give 1 ng STR Peaks Detected (out of 28) Intact / /28 2 u / /28 3 u / /28 4 u / /28 5 u / /28 6 u / / Applied Biosystems

48 Conclusions The Quantifiler Duo kit is a multiplex real time PCR assay for simultaneous quantification of total human and human male DNA IPC enables monitoring for the presence of PCR inhibitors The assay is specific for human and higher primates Provides information about the mixture ratio of total human and human male DNA in a sample High dynamic range of quantification from 23 pg/µl to 50 ng/µl The quantification results correlate well with Identifiler and YFiler kit performance Applied Biosystems

49 Acknowledgements Applied Markets Research and Development Jai Shewale, Maura Barbisin, Pius Brzoska, Robert Green, Michael Malicdem, Robert Lagace, Rixun Fang, Cristin O Shea, Julio Mulero, Lori Hennessy, Chien-Wei Chang and Manohar Furtado Applied Markets Marketing Heidi Kijenski, Karen Cormier and Lisa Calandro Applied Markets Product Development: Lindy Kauffman, Yixin N Li, Sabine Short and Beth Ladin Applied Biosystems

50 Legal Statements For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. Purchase of the Quantifiler Duo Kit includes an immunity from suit under patents specified in the product insert to use only the amount purchased solely in forensic and paternity testing, and also for the purchaser s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Not for re-sale. Applera, Applied Biosystems, AB (Design), AmpFlSTR, FAM, Identifiler, NED, Quantifiler, and Y-Filer are registered trademarks, and MiniFiler is a trademark of the Applera Corporation or its subsidiaries in the US and/or certain other countries Applied Biosystems

51 Thank You Quantifiler Duo DNA Quantification Kit