SNP and STR Multiplexes for NGS · 2020. 5. 5. · Applied Biosystems HID Ion AmpliSeq Ancestry and...
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AAFS 2016 Workshop: Considerations for Implementing NGS Technologies in a Forensic Laboratory 2/23/2016 1 Katherine B. Gettings, Ph.D. Research Biologist, Applied Genetics Group Workshop: Considerations for Implementing NGS Technologies Into a Forensic Laboratory 68 th Annual AAFS Meeting February 23, 2016 Las Vegas, NV SNP and STR Multiplexes for NGS Updated Slides: http://www.slideshare.net/NIST_AppliedGeneticsGroup Updated Slides: http://www.slideshare.net/NIST_AppliedGeneticsGroup SNPs and STRs by NGS 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 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 + FGx + PGM 83 IISNPs FGx Only 11 PGM Only 7 AISNPs FGx + 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 NGS of Forensic STR Loci Information
Transcript of SNP and STR Multiplexes for NGS · 2020. 5. 5. · Applied Biosystems HID Ion AmpliSeq Ancestry and...
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AAFS 2016 Workshop: Considerations for Implementing NGS Technologies in a Forensic Laboratory
2/23/2016
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Katherine B. Gettings, Ph.D.Research Biologist, Applied Genetics Group
Workshop: Considerations for Implementing NGS Technologies Into a Forensic Laboratory68th Annual AAFS MeetingFebruary 23, 2016Las Vegas, NV
SNP and STR Multiplexes for NGS
Updated Slides:http://www.slideshare.net/NIST_AppliedGeneticsGroup
Updated Slides:http://www.slideshare.net/NIST_AppliedGeneticsGroup
SNPs and STRsby NGS
Promega PowerSeq Auto/YSTR/Mito System
Applied Biosystems HID Ion AmpliSeqAncestry and Identity Panels
NOW AVAILABLE!!! Forensic NGS Productsfor STR and SNP Typing
Illumina ForenSeqand FGx
Assay Number of Samples in Run Estimated Cost per SampleLife Tech AmpliSeq SNP Panel (PGM) 6 $174
30 $122
59 $115
IlluminaForenSeq(FGx)
8 $215
32 $84
96 $55
PromegaPowerSeqAuto/Y/mito
8 $249
32 $110
96 $68
Let’s Make a Deal!estimates do not include instruments, labor or consumables
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FGx + PGM83 IISNPs
FGx Only11
PGM Only7
AISNPsFGx + PGM55
FGx Only1
PGM Only110
PGM 34
PISNPs
Y SNPs
FGx24*
FGx7
auSTRs
X STRs
Y STRs FGx + Promega20
Promega Only3
FGx Only6
FGx + Promega
22
FGx Only6
*2 FGx PISNPsoverlap with AISNPs
mtDNA Control RegionPromega
NGS of Forensic STR LociInformation
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[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
N=183
Length
Forensic STR Sequence Diversity
N=183
Simple Repeats
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
D12S391
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How NGS Helps in Mixtures D12S391 1:9:9 Mixture
PERSON 1
PERSON 2STUTTER FROM PERSON 2
PERSON 3STUTTER FROM PERSON 3
0
200
400
600
800
1000
1200
1400
1600
1800
2000
17 18 18.3 19 22 23 24
Coverag
e
Sequences > 75X Coverage
1 additional allele2 alleles
help with stutter
1 additional allele2 alleles
help with stutter
NGS of Forensic STR LociAssay Performance
ForenSeq – Primer Mix A27 auSTRs + 24 YSTRs + 7 XSTRs
+ 94 iiSNPs + 78 ai/piSNPs + Amel = 231
PowerSeq Auto + Y 22 auSTRs + 23 YSTRs + Amel = 46
PowerPlex Fusion22 auSTRs +
1 YSTR + Amel = 24
Number of Loci in Multiplex:
~50‐fold difference between lowest and highest coverage locus
Lowest locus = 100XHighest locus = 5000X
3.6‐fold difference between lowest and highest coverage locus
2‐fold difference between lowest and highest coverage locus
PowerSeq Auto22 auSTRs +
1 YSTR + Amel = 24
ForenSeq and Promega PowerSeq STR Size Range
ForenSeq auSTR ForenSeq X/YSTR Powerseq auSTR Powerseq YSTR85 – 467 102 – 462 129 – 264 139-294
Amplicon Size Range (bp):
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 STRsAutosomal STRs
*
XSTRs YSTRs
Heterozygote Balance
Coverage
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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 STRsAutosomal 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
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
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
NGS of Forensic SNP LociInformation
• IISNP‐Individual Identification SNP• AISNP‐Ancestry Informative SNP• PISNP‐Phenotype Informative SNP
SNP Information SNP Information
• IISNP‐Individual Identification• PGM Identity SNP Panel and ForenSeq both contain:– Kidd 45 and SNPforID52 – With occasional exceptions
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IISNP Benefits for Degraded DNA • PISNP‐Phenotype
• ForenSeq contains all 24 HIrisplex SNPs• Only NGS assay with phenotype SNPs
SNP Information
• AISNP ‐ Ancestry Information• PGM Ancestry SNP Panel contains
– Seldin 128 (with some exceptions)– Kidd 55
• ForenSeq contains Kidd 55
SNP Information Kosoy 2008 “Seldin 128”
Present in PGM Ancestry Panel
Kidd 2014“Kidd 55”
Present in ForenSeq and
PGM Ancestry Panel
NGS of Forensic SNP LociAssay Performance
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
Heterozygote Balance
Coverage
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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
Heterozygote Balance
Coverage
– 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
Ion Torrent PGM AISNP PanelForenSeq software uses Hirisplex Model for Phenotype Prediction
ForenSeq AISNP + PISNP Panel
And 1000 Genome data for Continental level ancestry prediction
PGM – Kidd 55
PGM – Seldin 128
ForenSeq – Kidd 55
African American IndividualPGM – Kidd 55
PGM – Seldin 128
ForenSeq – Kidd 55
East Asian Individual
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PGM – Kidd 55
PGM – Seldin 128
ForenSeq – Kidd 55
Half European –Half East Asian Individual
PGM – Kidd 55
PGM – Seldin 128
ForenSeq – Kidd 55
Hispanic Individual
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
Individuals Grouped by Self Described Eye ColorBrown Blue
Hirisplex Eye Color Prediction
Categories
Individuals Grouped by Self Described Hair ColorBlack Very Dark Brown Dark Brown Medium Brown Light Brown
Hirisplex Hair Color Prediction Categories
NGS of SNPs ‐ Conclusions• 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
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Acknowledgements
?
NISTPete ValloneKevin KieslerLisa BorsukNate Olson
Becky SteffenMargaret Kline
Mike CobleDave Duewer
GWUDaniele PodiniRachel Aponte
Acknowledgements
Contact Information [email protected]
NIST Disclaimer: Certain commercial equipment, instruments and materials areidentified in order to specify experimental procedures as completely as possible.In no case does such identification imply a recommendation or it imply that anyof the materials, instruments or equipment identified are necessarily the bestavailable for the purpose. Funding FBI: DNA as a Biometric
