Validation Studies on DNA Mixtures Using the PowerPlex Fusion 6C System

Transcription

1 Promega Webinars eries: Genetic Identity INAEX: Improving NA ata Exchange Validation tudies on NA Mixtures Using the PowerPlex Fusion 6C ystem INTCF-Madrid ERVICIO E BIOLOGIA upported by a grant from the IEC programe //

2 Proposal: Validation of a single TR typing system based on 6-ye CE detection to improve NA data Exchange locally and globally IEC Programme Targeted Call Awarded July Requirements to the Companies TR covering E & COI +, & E Concordance of primer sequences design with previous systems Master mix with high tolerance to PCR inhibitor for casework application E TR Core Loci COI TR Core Loci Other TR Loci In Forensic Kits panish atabase (. profiles) Year CFPO TPOX TR Markers in E typing kits CFPO TPOX FGA FGA FGA FGA TH TH TH TH VWA VWA VWA VWA E Penta Penta E E Proposed TR Typing ystem CFPO TPOX 6 FGA TH VWA 66 E

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4 General characteristics of TR markers included in the Promega 6 ye Kit (V) and forensic biostatistical parameters in the panish population (N=) Garcia et al. Forensic ci Int Genet. 6() () Marker Chromosomal Location Basic Repeat motif ye Label Allele sizes (bp) He P CE Y Promega 6C V 6 AMEL X: p.-. ; Y: p. -- FL-6C, p. TCTA/TCTG q. TAGA p TCTA q6. GGAA q- TATC Penta E q6. AAAGA q. GATA JOE-6C q. AGAA q TGCC/TTCC CFPO q.- AGAT Penta q. AAAGA -... TH p. AATG/ATG TMR-6C -... vwa p. TCTA/TCTG -... q.-q TCTA/TCTG q.- GATA q- AGAT TPOX p-per AATG -... q. TCTA/TCTG CXR-6C p. AGAT/AGAC q AAGG E 6q Complex AAAG -... q. ATT Y Yq. TCTA TOM-6C FGA q CTTT Y6 Yq AAAG Y Yq TTTC

5 Validation Parameters & Materials Validation Parameters Analytical Threshold ensitivity & tochastic Threshold Precision and Accuracy Repeatability & reproducibility Heterozygous balance tutter Threshold Genotype concordance NA mixtures pecies specificity PCR reduced reaction volume tability study in Case-Type amples PCR negative controls Type & Number of amples Analyzed Human genomic NA samples of known human NA concentration (RM-A, NA Control & genomic NA sample from an anonymous male donor) ( samples: Quadruplicate or Triplicate analysis of -6 dilution series) injections of the Globalfiler allelic ladder human genomic NA samples Human genomics NA samples of known TR profile ( samples: replicates of samples generated by two operators) Human genomic NA samples Human genomic NA samples Human genomic NA samples from saliva swabs of anonymous donors genomic NA samples of the NA profiling standard RM-b male /female NA mixture in different ratios ( ng of total NA in triplicate) male /female NA mixture in different ratios ( ng of total NA in triplicate) ( samples: Triplicate analysis of dilutions from series) NA mixture samples from proficiency exercises genomic NA from human associated microbial species human genomic NA samples of known TR profile forensic NA samples previously analyzed with different TR Kits

6 Methods NA Extraction NA Quantification Real time PCR Quantifiler uo Kit Targets: RPPH, RY & IPC Reference samples Forensic samples (ifferential Lysis for emen) PCR Amplification X 6ºC for 6 s X 6ºC for s, 6-6ºC for 6 s X 6ºC for min CE etection ABI ample Inyection: s at.kv CE: s at kv ata Analysis GM-IX Promega 6 ye Manual Peak Amplitude RFU Management of samples, analysis and results Comparison & Concordance studies

7 Promega 6 ye TR profile from a reference saliva swab (NA input: pg; PCR Reaction volume :. ul)

8 Analytical Threshold : PCR negative controls Range of analysis: 6- bp; Peak etector: RFU Promega 6C V IL Promega 6C V- IL WEN Promega 6C V Blue Green Yellow Red Purple Average RFU,,,,,6 tandard deviation,,,,, Max. RFU 6 6 Min. RFU Average RFU + tandard deviation,6 6,,,, Average RFU + tandard deviation, 6, 6,,, X (Ymax - Ymin) AT: RFU AT: RFU Promega 6C V Blue Green Yellow Red Purple Average RFU,,, 6,, tandard deviation,, 6,, 6, Max. RFU Min. RFU Average RFU + tandard deviation,,,,, Average RFU + tandard deviation,6 6, 6,, 6, AT: RFU X (Ymax - Ymin) 6 AT: RFU

9 ensitivity study & stochastic threshold (Promega 6C V) Heat maps summarizing the sensitivity results of the last five NA serial dilutions (from to. pg) A M A M E L 6 6 P e n t a E Y C F P O P e n t a tochastic threshold RFU value of the highest surviving false homozygous peak when allele drop-out is observed at heterozygous markers: 6 RFU for at 6. pg of NA input T H v W A T P O X E 6 F G A Y 6 Y NA Input (pg) Observed ropout % % 6 % % % NA Input (pg) Observed ropout % % 6 % % 6 %

10 ensitivity study & stochastic threshold(promega 6C V) Heat maps summarizing the sensitivity results of five NA serial dilutions (from to pg) tochastic threshold RFU value of the highest surviving false homozygous peak when allele drop-out is observed at heterozygous markers: RFU for at 6. pg of NA input A M E L 6 6 P e n t a E 6 C F P O P e n t a T H v W A T P O X E Y F G A Y 6 Y GENOMIC NA (NA) NA Input (pg) Observed ropout % % % 6 % 6 %

11 ensitivity study: Incidence of rop-in at Low NA input -A- PG E-A-6 PG FGA-M-6 PG 6 RFU RFU RFU -NA-6 PG FGA-NA- PG etected Incidence of rop-in for the last four dilution series over a threshold of RFU (, eterminations) RFU RFU RFU. %

12 NA mixtures series: Relative ratios of :, :, :, :, :, :, and : of female / male A M E L 6 6 P e n t a E Y C F P O P e n t a T H v W A T P O X E 6 F G A Y 6 Y TOTAL NA INPUT: ng : : : : : : : : - : - : - 666: - 666: - 666: - :66 - :66 - :66 - : - : - : - : - : - : - :6 - :6 - :6 - : - : - : - TOTAL NA INPUT: ng : : : : : : : : - : - : - : - : - : - : - : - : - 66: - 66: - 66: - : - : - : - :6 - :6 - :6-6: - 6: - 6: -

13 Full profile of the minor male contributor: eries of ng of total NA at : mixture ratio NA input of the minor male contributor: pg

14 Full profile of the minor male contributor: eries of ng of total NA at : mixture ratio High Pull up & High tutter Peaks NA input of the minor male contributor: 6 pg

15 Body fluid mixture samples from GHEP & GENAP proficiency exercises Two Contributors ALIVA NA AMPLE BOY FLUI & CONTRIBUTOR [NA] ng/ul NAMIX-L Total:. Male:. NAMIX-L Total:. Male:. NAMIX-L Total:. Male:.6 NAMIX-L Total: Male: NAMIX Total: Male: NAMIX Total: Male: NAMIX Total: Male: NAMIX6 Total:. Male: NAMIX Total:. Male:. NAMIX Total:. Male:. NAMIX Total:. Male:. NAMIX Total: Male: EMEN BLOO Three Contributors NA AMPLE BOY FLUI & CONTRIBUTOR [NA] ng/ul NAMIX Total: Male: NAMIX Total:.6 Male:. NAMIX Total:. Male:. NAMIX Total:. Male:. Allele Concordance with other kits MNC MNM

16 Allele Concordance with other Kits (Intra & Inter-lab comparison) NA AMPLE NAMIX-L NAMIX-L BOY FLUI & CONTRIBUTOR Allele Concordance (Globalfiler, Identifiler, NGME, EX, ) Partial NA profile from the minor male contributor at a : NA ratio NAMIX-L NAMIX Two Contributors NAMIX NAMIX NAMIX NAMIX6 Non-concordance at E among different Kits (primer designs) Allele at (Out of ladder) NAMIX NAMIX NAMIX NAMIX

17 Allele Concordance with other Kits (Intra & Inter-lab comparisons) Three Contributors NA AMPLE NAMIX NAMIX NAMIX NAMIX BOY FLUI & CONTRIBUTOR Allele Concordance (Globalfiler, Identifiler, NGME, EX, ) CE Resolution of Microvariants at CE Resolution of Microvariants at

18 Partial NA profile from the minor male contributor (Female:male mixture ratio :) PENTA E PENTA E PENTA PENTA FGA FGA Y6 Y Y6 Y

19 Non-concordance at E among different Kits (primer designs) E.. Promega 6C V E.. Globalfiler

20 Allele at (Out of ladder) Promega 6C V Globalfiler

21 CE Resolution of Microvariants at Promega 6C V Automatic allele calling of. By GMIX reanalysis with: No moothing. Globalfiler

22 NA AMPLE Minimum Number of Contributors BOY FLUI & CONTRIBUTOR Total of Alleles Minimum Number of Contributors TR with alleles TR with alleles NAMIX-L 6 MNC NAMIX-L 6 NAMIX 6 NAMIX NAMIX NAMIX NAMIX6 6 6 NAMIX 6 6 NAMIX 6 6 NAMIX NAMIX

23 Minimum Number of Contributors NA AMPLE BOY FLUI & CONTRIBUTOR Minimum Number of Contributors Total of Alleles TR with alleles TR with 6 alleles NAMIX MNC NAMIX NAMIX NAMIX

24 Minimum Number of Males? Y Y6 Y MNM NAMIX NAMIX-L NAMIX NAMIX NAMIX

25 Concluding Remarks The data reported demonstrated the Promega 6C PCR amplification system followed by CE detection in a genetic analyzer generates high quality, reproducible, precise, accurate, and sensitive profiling TR data, even from subnanogram amounts of genomic NA template. The study demonstrated that the combination of autosomal TRs and Y-TRs in the PowerPlex 6-dye system maximizes the information obtained from NA mixtures regarding both the minimum number of total contributors and the minimum number of specific male contributors. The Promega 6C system provides equivalent overall performance to previous forensic TR PCR kits, but with enhanced discrimination power for a better match efficiency that would reduce the chance of adventitious matches during NA data exchange among national NA databases

26 Concluding Remarks Multiplex TR ystem Probability of Identity Probability of Paternity Exclusion NGMElect. X -.% Globalfiler. X -6.% Promega 6C ystem. X -.% (panish population. Garcia et al. Forensic ci Int Genet. 6() ()) The use of Promega 6C ystem would be also very beneficial to improve discrimination power of NA analysis not only in criminal NA databases, but also in many other forensic applications of autosomal TR profiling including missing person identification, disaster victim identification, mass grave investigations and kinship analysis.

27 Acknowledgements ouglas torts & The Promega Team Kit esign & Production Oscar Garcia Biostatistical Calculation with the panish Population ata PROJECT TEAM Pablo Martín Lourdes Fernández de imón Gracia Luque Mª José Farfán Antonio Alonso INTCF-Madrid ERVICIO E BIOLOGIA BIOLOGY EPARTMENT TAFF This project has been funded with support from the European Commission (grant HOME//IEC/AG/PRUM/ under the Prevention of and Fight against Crime (IEC) programme of the European Commission-irectorate General Justice and Home Affairs). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein.