![[TCTA][TCTG]2[TCTA]12
[TCTA][TCTG][TCTA]13
Sequence-Based Heterozygote or Isoallele:
A locus that appears homozygous
by length-based measurements (CE),
but is heterozygous by sequence
Additional Alleles by Sequence at STR Loci
NIST Applied Genetics Group
NIST Applied Genetics Group
NIST Applied Genetics Group](https://image.slidesharecdn.com/aafsngs-workshopgettings-160219225929/85/SNP-and-STR-Multiplexes-for-NGS-7-320.jpg)
SNP and STR Multiplexes for NGS
- 1. Katherine B. Gettings, Ph.D. Research Biologist, Applied Genetics Group Workshop: Considerations for Implementing NGS Technologies Into a Forensic Laboratory 68th Annual AAFS Meeting February 23, 2016 Las Vegas, NV SNP and STR Multiplexes for NGS Updated Slides: http://www.slideshare.net/NIST_AppliedGeneticsGroup NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 2. Updated Slides: http://www.slideshare.net/NIST_AppliedGeneticsGroup SNPs and STRs by NGS NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 3. Promega PowerSeq Auto/YSTR/Mito System Applied Biosystems HID Ion AmpliSeq Ancestry and Identity Panels NOW AVAILABLE!!! Forensic NGS Products for STR and SNP Typing Illumina ForenSeq and FGx NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 4. Assay Number of Samples in Run Estimated Cost per Sample Life Tech AmpliSeq SNP Panel (PGM) 6 $174 30 $122 59 $115 Illumina ForenSeq (FGx) 8 $215 32 $84 96 $55 Promega PowerSeq Auto/Y/mito 8 $249 32 $110 96 $68 Let’s Make a Deal! estimates do not include instruments, labor or consumables + NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 5. FGx + PGM 83 IISNPs FGx Only 11 PGM Only 7 AISNPsFGx + PGM 55 FGx Only 1 PGM Only 110 PGM 34 PISNPs Y SNPs FGx 24* FGx 7 auSTRs X STRs Y STRs FGx + Promega 20 Promega Only 3 FGx Only 6 FGx + Promega 22 FGx Only 6 *2 FGx PISNPs overlap with AISNPs mtDNA Control Region Promega NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 6. NGS of Forensic STR Loci Information NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 7. [TCTA][TCTG]2[TCTA]12 [TCTA][TCTG][TCTA]13 Sequence-Based Heterozygote or Isoallele: A locus that appears homozygous by length-based measurements (CE), but is heterozygous by sequence Additional Alleles by Sequence at STR Loci NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 8. N=183 Length NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 9. Forensic STR Sequence Diversity N=183 Simple RepeatsNIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 10. NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 11. NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 12. How NGS “Helps” with Stutter AAFS Criminalistics Session B180 - Friday Morning Aponte et al. Sequence-Based Analysis of Stutter at STR Loci: Implementation and Utilization D12S391NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 13. How NGS Helps in Mixtures NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 14. D12S391 1:9:9 Mixture PERSON 1 PERSON 2 STUTTER FROM PERSON 2 PERSON 3 STUTTER FROM PERSON 3 0 200 400 600 800 1000 1200 1400 1600 1800 2000 17 18 18.3 19 22 23 24 Coverage Sequences > 75X Coverage 1 additional allele 2 alleles help with stutter NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 15. NGS of Forensic STR Loci Assay Performance NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 16. ForenSeq – Primer Mix A 27 auSTRs + 24 YSTRs + 7 XSTRs + 94 iiSNPs + 78 ai/piSNPs + Amel = 231 PowerSeq Auto + Y 22 auSTRs + 23 YSTRs + Amel = 46 PowerPlex Fusion 22 auSTRs + 1 YSTR + Amel = 24 Number of Loci in Multiplex: ~50-fold difference between lowest and highest coverage locus Lowest locus = 100X Highest locus = 5000X 3.6-fold difference between lowest and highest coverage locus 2-fold difference between lowest and highest coverage locus PowerSeq Auto 22 auSTRs + 1 YSTR + Amel = 24 NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 17. ForenSeq and Promega PowerSeq STR Size Range ForenSeq auSTR ForenSeq X/YSTR Powerseq auSTR Powerseq YSTR 85 – 467 102 – 462 129 – 264 139-294 Amplicon Size Range (bp): NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 18. 32 sample run 27 autosomal STRs + Amelogenin Left side y-axis = heterozygote balance • ranges from 0.60 to 0.89 Right side y-axis = average STR coverage • ranges from 133X to 7332X 7 XSTRs - average coverage ranges from • 140X to 2807X for males and • 299X to 5463X for females 24 YSTRs - average coverage ranges from • 118X to 5628X ForenSeq STRs Autosomal STRs * XSTRs YSTRs NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 19. 32 sample run 27 autosomal STRs + Amelogenin Left side y-axis = heterozygote balance • ranges from 0.60 to 0.89 Right side y-axis = average STR coverage • ranges from 133X to 7332X ForenSeq and PowerSeq STRs Autosomal STRs 96 sample run 22 autosomal STRs + 1 YSTR + Amelogenin Left side y-axis = heterozygote balance • ranges from 0.87 to 0.91 Right side y-axis = average STR coverage • ranges from 3100X to 5900X NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 20. ForenSeq STR and IISNP Statistics Length-based allele frequencies Green Bar = sample result Gray Bar = scaled to range in population Gray Line = source attribution threshold NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 21. NGS of STRs - Conclusions • Two assays are available for sequencing forensic STR loci • STR sequencing will increase allelic diversity, improving differentiation among individuals in a mixture – Locus specific gains in repeat regions and flanking regions – Extent of gain is difficult to quantify • Characterize “peak height ratios”, interlocus balance and stutter by NGS (assay and locus specific) • Sequence-based allele frequency databases NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 22. NGS of Forensic SNP Loci Information NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 23. • IISNP-Individual Identification SNP • AISNP-Ancestry Informative SNP • PISNP-Phenotype Informative SNP SNP Information NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 24. SNP Information • IISNP-Individual Identification • PGM Identity SNP Panel and ForenSeq both contain: – Kidd 45 and SNPforID52 – With occasional exceptions NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 25. IISNP Benefits for Degraded DNA NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 26. • PISNP-Phenotype • ForenSeq contains all 24 HIrisplex SNPs • Only NGS assay with phenotype SNPs SNP Information NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 27. • AISNP - Ancestry Information • PGM Ancestry SNP Panel contains – Seldin 128 (with some exceptions) – Kidd 55 • ForenSeq contains Kidd 55 SNP Information NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 28. Kosoy 2008 “Seldin 128” Present in PGM Ancestry Panel Kidd 2014 “Kidd 55” Present in ForenSeq and PGM Ancestry Panel NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 29. NGS of Forensic SNP Loci Assay Performance NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 30. 172 SNPs - Identity, Ancestry & Phenotype Contains (with a few exceptions): • Kidd 55 for Ancestry • Kidd 45 + SNPforID52 for Identity • HIrisplex 24 Left side y-axis = average SNP coverage • ranges from 23X to 3567X (>150 fold) Right side y-axis = average het balance • ranges from 0.42 to 0.94 ForenSeq SNPs NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 31. Ion Torrent PGM AISNP Panel 165 SNPs - Ancestry Contains Kidd 55 and Seldin 128 (with exceptions) Left side y-axis = average SNP coverage • ranges from 264X to 2000X (7.6 fold) Right side y-axis = average het balance • ranges from 0.43 to 0.98 NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 32. – PGM plugin uses ALFRED population data • Evaluates expected occurrence of profile in each ALFRED population • Potential for very specific ancestry prediction Ion Torrent PGM AISNP Panel NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 33. Ion Torrent PGM AISNP Panel NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 34. ForenSeq software uses Hirisplex Model for Phenotype Prediction ForenSeq AISNP + PISNP Panel And 1000 Genome data for Continental level ancestry prediction NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 35. PGM – Kidd 55 PGM – Seldin 128 ForenSeq – Kidd 55 African American Individual NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 36. PGM – Kidd 55 PGM – Seldin 128 ForenSeq – Kidd 55 East Asian Individual NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 37. PGM – Kidd 55 PGM – Seldin 128 ForenSeq – Kidd 55 Half European – Half East Asian Individual NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 38. PGM – Kidd 55 PGM – Seldin 128 ForenSeq – Kidd 55 Hispanic Individual NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 39. PGM – Kidd 55 Hispanic Individual Equally likely to be Central Asian, Native American or European Over a billion times more likely European / Central Asian / Native American than East Asian / African NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 40. Individuals Grouped by Self Described Eye Color Brown Blue Hirisplex Eye Color Prediction Categories NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 41. Individuals Grouped by Self Described Hair Color Black Very Dark Brown Dark Brown Medium Brown Light Brown Hirisplex Hair Color Prediction Categories NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 42. NGS of SNPs - Conclusions • Ancestry SNP prediction relies on appropriate training data • More guidance is needed for ancestry and phenotype prediction interpretation • Identity panels may be useful in degraded samples • Combining across SNP panels and with STR data may be useful – LD must be evaluated NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 43. Acknowledgements NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group
- 44. NIST Pete Vallone Kevin Kiesler Lisa Borsuk Nate Olson Becky Steffen Margaret Kline Mike Coble Dave Duewer GWU Daniele Podini Rachel Aponte Acknowledgements Contact Information katherine.gettings@nist.gov NIST Disclaimer: Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose. Funding FBI: DNA as a Biometric NIST Applied Genetics Group NIST Applied Genetics Group NIST Applied Genetics Group