Building a large-scale missing persons ID SNP panel - Download the study
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In this webinar, we will take a look at a large-scale SNP-based forensic identification panel for DNA analysis with massively parallel sequencing (MPS). The panel was specifically designed for the challenges of identifying missing persons; where DNA is frequently highly degraded, and relationship tests may involve reference samples from across several generations and in a deficient pedigree.
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Building a large-scale missing persons ID SNP panel - Download the study
- 1. Building a large-scale missing persons ID SNP panel Christopher Phillips A. Tillmar, T.J. Parsons, R. Huel, K. Kidd, M.V. Lareu, K. Elliott, R. Samara, E. Lader Forensic Genetics Unit, University of Santiago de Compostela, Spain
- 2. Building a large-scale missing persons ID SNP panel Christopher Phillips A. Tillmar, T.J. Parsons, R. Huel, K. Kidd, M.V. Lareu, K. Elliott, R. Samara, E. Lader Forensic Genetics Unit, University of Santiago de Compostela, Spain Yale
- 3. ICMP 2.0 aims to redefine the scope of its identification work worldwide and adopt MPS as key technology, founded in the new headquarters The Hague facilities will be well suited to adoption of new forensic DNA technologies Bioinformatics and computational infrastructure is well founded Qiagen and ICMP have agreed to develop a complete missing persons ID pipeline
- 4. The rationale for SNP analysis in forensics Microhaplotype genotyping becomes possible with the introduction of MPS Yale
- 5. The rationale for SNP analysis in forensics Microhaplotype genotyping becomes possible with the introduction of MPS Yale Constructed a new MPS panel dedicated to missing persons identification consisting of multiple- allele SNPs and microhaplotypes A collaborative project that makes use of the QIAseq MPS system: originally developed for sensitivity to low-level mutation sequences
- 6. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel
- 7. Single Nucleotide Polymorphisms can provide complimentary data to STRs SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Yale
- 8. Single Nucleotide Polymorphisms can provide complimentary data to STRs SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Yale
- 9. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs Yale Single Nucleotide Polymorphisms can provide complimentary data to STRs
- 10. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs Yale Single Nucleotide Polymorphisms can provide complimentary data to STRs
- 11. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs Yale The amplified fragments used to genotype SNPs can be very short SNPs can be more successful than STRs when the DNA is extremely degraded Single Nucleotide Polymorphisms can provide alternative tests to STRs
- 12. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs Yale The amplified fragments used to genotype SNPs can be very short SNPs can be more successful than STRs when the DNA is extremely degraded Single Nucleotide Polymorphisms can provide alternative tests to STRs
- 13. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs Yale The amplified fragments used to genotype SNPs can be very short SNPforID 52-plex SNP panel Innsbruck experimental Mini-STRs NIST NC-01/NC-02 Mini-STRs SNPs can be more successful than STRs when the DNA is extremely degraded Single Nucleotide Polymorphisms can provide alternative tests to STRs
- 14. Yale • Bode technologies used Orchid’s Snippet system to genotype small-scale SNP multiplexes
- 15. Yale • Bode technologies used Orchid’s Snippet system to genotype small-scale SNP multiplexes • Compared to mtDNA analysis this early SNP typing pilot study was largely unsuccessful
- 16. Single Nucleotide Polymorphisms are one form of short genetic variation SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs The amplified fragments used to genotype SNPs can be very short Yale SNPs can be more successful than STRs when the DNA is extremely degraded
- 17. The amplified fragments used to genotype SNPs can be very short SNPs in coding regions underlie a large proportion of common genetic variation SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs Yale But new types of marker can now be genotyped with MPS SNPs can be more successful than STRs when the DNA is extremely degraded
- 18. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs The amplified fragments used to genotype SNPs can be very short SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power New types of SNP-based markers But new types of marker can now be genotyped with MPS SNPs can be more successful than STRs when the DNA is extremely degraded
- 19. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs The amplified fragments used to genotype SNPs can be very short SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power New types of SNP-based markers But new types of marker can now be genotyped with MPS SNPs can be more successful than STRs when the DNA is extremely degraded
- 20. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs T G C A T A The amplified fragments used to genotype SNPs can be very short SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power New types of SNP-based markers But new types of marker can now be genotyped with MPS SNPs can be more successful than STRs when the DNA is extremely degraded
- 21. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs T G C A T A The amplified fragments used to genotype SNPs can be very short SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power New types of SNP-based markers But new types of marker can now be genotyped with MPS SNPs can be more successful than STRs when the DNA is extremely degraded
- 22. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs T G C A T A Microhaplotypes SNPs in and around STRs The amplified fragments used to genotype SNPs can be very short SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power New types of SNP-based markers But new types of marker can now be genotyped with MPS SNPs can be more successful than STRs when the DNA is extremely degraded
- 23. SNPs in coding regions underlie a large proportion of common genetic variation SNPs form the basis of forensic trait-predictive tests of externally visible characteristics (EVCs) and much of the ancestry informative markers in forensic ancestry tests Ancestry Informative SNPsTrait-Predictive SNPs Identity Testing SNPs Lineage SNPs T G C A T A Microhaplotypes SNPs in and around STRs Both can now be genotyped with MPS The amplified fragments used to genotype SNPs can be very short SNPs in closely-linked sets can be jointly genotyped by Massively Parallel Sequencing techniques Phased microhaplotypes have multiple alleles - increasing their discrimination power New types of SNP-based markers SNPs can be more successful than STRs when the DNA is extremely degraded But new types of marker can now be genotyped with MPS
- 24. • Short amplicons give best results even with low quants • SNP dropout is random rather than systematic SNPs work well with very degraded DNA
- 25. • SNP dropout is random rather than systematic No.ofreportableloci(46intotal) DNAconcentrationng/μl • Short amplicons give best results even with low quants SNPs work well with very degraded DNA
- 26. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel
- 27. Adaptor Barcode + Adaptor Barcode + Adaptor Adaptor Clonal amplification by Bridge Amplification Add adaptors and barcodes Clean up Library Removes excess reagents Barcode + Adaptor Adaptor Repair ends and add A overhang Adaptor Barcode + Adaptor Add adaptors and barcodes Clean up Library Removes excess reagents Barcode + Adaptor Adaptor Clonal amplification by Bridge Amplification Create blunt ends Adaptor Barcode + Adaptor Clonal amplification by Emulsion PCR DNA fragments bind to Ion Sphere Particles (ISPs) I S P I S P D Target amplification DNA is replicated and new fragments bind to ISPs Removes oil and excess reagents from PCR Clean up Clean up Cluster generation CLONAL AMPLIFICATION SEQUENCING LIBRARY PREPARATION Hybridize fragments to flow cell glass surface Single-stranded DNA flexes to form a bridge, then extended by polymerase Double-stranded bridge denatured leaving two single-stranded fragments Process repeated to form cluster of fragments Reverse strands cleaved and washed off to leave forward strand clusters on flow cell Spheres loaded into individual wells on surface of semi- conductor chip Chip surface is flushed and drained with successive dNTPs Voltage spikes converted to Ionogram Nucleotide addition causes a change in pH with proportional voltage charge Sensor plate Sensing layer Sequencing primers added Sequencing performed on forward strands. As dNTPs are added fluorescence is emitted as light signals measured by a detector Regenerate DNA fragments for reverse strand on flow cell Sequencing is repeated on reverse strands. Light signals converted to sequence read PCR AMPLIFICATIONDNA EXTRACTION QUANTITATION Replicate DNA with forensically relevant primer sets to target specific sitesMeasure amount of DNARelease DNA from cells STANDARD FORENSIC TARGET DNA PREPARATION Dr. Linzi Wilson-Wilde MPS
- 28. Adaptor Barcode + Adaptor Barcode + Adaptor Adaptor Clonal amplification by Bridge Amplification Add adaptors and barcodes Clean up Library Removes excess reagents Barcode + Adaptor Adaptor Repair ends and add A overhang Adaptor Barcode + Adaptor Add adaptors and barcodes Clean up Library Removes excess reagents Barcode + Adaptor Adaptor Clonal amplification by Bridge Amplification Create blunt ends Adaptor Barcode + Adaptor Clonal amplification by Emulsion PCR DNA fragments bind to Ion Sphere Particles (ISPs) I S P I S P D Target amplification DNA is replicated and new fragments bind to ISPs Removes oil and excess reagents from PCR Clean up Clean up Cluster generation CLONAL AMPLIFICATION SEQUENCING LIBRARY PREPARATION Hybridize fragments to flow cell glass surface Single-stranded DNA flexes to form a bridge, then extended by polymerase Double-stranded bridge denatured leaving two single-stranded fragments Process repeated to form cluster of fragments Reverse strands cleaved and washed off to leave forward strand clusters on flow cell Spheres loaded into individual wells on surface of semi- conductor chip Chip surface is flushed and drained with successive dNTPs Voltage spikes converted to Ionogram Nucleotide addition causes a change in pH with proportional voltage charge Sensor plate Sensing layer Sequencing primers added Sequencing performed on forward strands. As dNTPs are added fluorescence is emitted as light signals measured by a detector Regenerate DNA fragments for reverse strand on flow cell Sequencing is repeated on reverse strands. Light signals converted to sequence read PCR AMPLIFICATIONDNA EXTRACTION QUANTITATION Replicate DNA with forensically relevant primer sets to target specific sitesMeasure amount of DNARelease DNA from cells STANDARD FORENSIC TARGET DNA PREPARATION MPS Dr. Linzi Wilson-Wilde
- 29. Mixed marker MPS panels like the Illumina DNA Signature Kit sequence the shortest possible amplicon lengths - here the bulk of SNPs are below 125 bp Yale STR fragment length ranges SNP fragment sizes
- 30. • Tested a DNA extract from a 12th Century male skeleton in duplicated MPS runs • Volders mediaeval burial site in Tyrol has 5th/6th Century skeletons overlaid with later 12th/13th Century remains A recent pilot study showed the sensitivity of MPS
- 31. N / NN QUAL=0 SNPs >100 x coverage SNPs 20-100 x coverage • Tested a DNA extract from a 12th Century male skeleton in duplicated MPS runs • Volders mediaeval burial site in Tyrol has 5th/6th Century skeletons overlaid with later 12th/13th Century remains A recent pilot study showed the sensitivity of MPS
- 32. 47 68 108 130 45 SNPs were removed from the prototype set 60 SNPs have reduced amplicon sizes compared to the prototype set (by an average 57.5 bp) 64 SNPs retain the original prototype primer designs 120 123 137 117 119 99 Prototype SNP panel amplicons Final HID-Ion AmpliSeq™ Identity Panel amplicons N / NN QUAL=0 SNPs >100 x coverage SNPs 20-100 x coverage
- 33. 47 68 108 130 45 SNPs were removed from the prototype set 60 SNPs have reduced amplicon sizes compared to the prototype set (by an average 57.5 bp) 64 SNPs retain the original prototype primer designs 120 123 137 117 119 99 Prototype SNP panel amplicons Final HID-Ion AmpliSeq™ Identity Panel amplicons N / NN QUAL=0 SNPs >100 x coverage SNPs 20-100 x coverage TFS Precision ID Identification SNPs Identification SNP Prototype Panel 169 124
- 34. Yale
- 35. Adaptor Barcode + Adaptor Barcode + Adaptor Adaptor Clonal amplification by Bridge Amplification Add adaptors and barcodes Clean up Library Removes excess reagents Barcode + Adaptor Adaptor Repair ends and add A overhang Adaptor Barcode + Adaptor Add adaptors and barcodes Clean up Library Removes excess reagents Barcode + Adaptor Adaptor Clonal amplification by Bridge Amplification Create blunt ends Adaptor Barcode + Adaptor Clonal amplification by Emulsion PCR DNA fragments bind to Ion Sphere Particles (ISPs) I S P I S P D Target amplification DNA is replicated and new fragments bind to ISPs Removes oil and excess reagents from PCR Clean up Clean up Cluster generation CLONAL AMPLIFICATION SEQUENCING LIBRARY PREPARATION Hybridize fragments to flow cell glass surface Single-stranded DNA flexes to form a bridge, then extended by polymerase Double-stranded bridge denatured leaving two single-stranded fragments Process repeated to form cluster of fragments Reverse strands cleaved and washed off to leave forward strand clusters on flow cell Spheres loaded into individual wells on surface of semi- conductor chip Chip surface is flushed and drained with successive dNTPs Voltage spikes converted to Ionogram Nucleotide addition causes a change in pH with proportional voltage charge Sensor plate Sensing layer Sequencing primers added Sequencing performed on forward strands. As dNTPs are added fluorescence is emitted as light signals measured by a detector Regenerate DNA fragments for reverse strand on flow cell Sequencing is repeated on reverse strands. Light signals converted to sequence read PCR AMPLIFICATIONDNA EXTRACTION QUANTITATION Replicate DNA with forensically relevant primer sets to target specific sitesMeasure amount of DNARelease DNA from cells Dr. Linzi Wilson-Wilde STANDARD FORENSIC TARGET DNA PREPARATION The PCR and library preparation steps build more complex oligos in the QIAseq chemistry
- 36. Yale The capture PCR used as the preamble to MPS can produce artefacts The capture PCR is prone to DNA synthesis errors (at a small frequency but exacerbated when analysing low-level DNA) Stochastic effects can be accelerated by big differences in GC content and therefore Tm values for any one DNA fragment
- 37. Yale 568 amplicons The capture PCR is prone to DNA synthesis errors (at a small frequency but exacerbated when analysing low-level DNA) PCR artefacts carrying through to MPS sequence output are particularly prevalent in the much larger multiplexes typically used in medical sequencing analyses The capture PCR used as the preamble to MPS can produce artefacts Stochastic effects can be accelerated by big differences in GC content and therefore Tm values for any one DNA fragment
- 38. DNA Fragmentation Library construction ligating Barcodes - Sample indices - F-primer sequences Sample indexing and amplification MPS-ready library MB Molecular Barcode GSP Gene-specific primer UP Universal primer FP Forward F-primer SIP Sample index primer ~9 hours Target enrichment by single primer extension of F-primer & Gene (SNP/MH) specific primer Ligation of extended oligos Yale QIAseq MPS chemistry combines a stepwise series of specific sequences into a composite oligonucleotide by ligation 568 amplicons
- 39. DNA Fragmentation Sample indexing and amplification MPS-ready library MB Molecular Barcode GSP Gene-specific primer UP Universal primer FP Forward F-primer SIP Sample index primer ~9 hours Target enrichment by single primer extension of F-primer & Gene (SNP/MH) specific primer Ligation of extended oligos Yale Library construction ligating Barcodes - Sample indices - F-primer sequences QIAseq MPS chemistry combines a stepwise series of specific sequences into a composite oligonucleotide by ligation
- 40. DNA Fragmentation Sample indexing and amplification MPS-ready library MB Molecular Barcode GSP Gene-specific primer UP Universal primer FP Forward F-primer SIP Sample index primer ~9 hours Target enrichment by single primer extension of F-primer & Gene (SNP/MH) specific primer Ligation of extended oligos Yale Library construction ligating Barcodes - Sample indices - F-primer sequences QIAseq MPS chemistry combines a stepwise series of specific sequences into a composite oligonucleotide by ligation Tag target DNA fragments with UMIs: Unique Molecular Indices Amplify Correct errors with UMIs
- 41. DNA Fragmentation Library construction ligating Barcodes - Sample indices - F-primer sequences Sample indexing and amplification MPS-ready library MB Molecular Barcode GSP Gene-specific primer UP Universal primer FP Forward F-primer SIP Sample index primer ~9 hours Target enrichment by single primer extension of F-primer & Gene (SNP/MH) specific primer Ligation of extended oligos A typical QIAseq oligo prepared for MiSeq analysis Yale QIAseq MPS chemistry combines a stepwise series of specific sequences into a composite oligonucleotide by ligation
- 42. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel
- 43. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel
- 44. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel • SNPs provide very short fragment PCR - markers of choice for degraded DNA • But SNPs clearly have much less information per marker than STRs • What happens when we use SNPs to examine a very distant kinship claim ?
- 45. Yale Second cousins in a fully deficient pedigree is a challenging kinship analysis to perform - in 2011 we opted to use an Affymetrix 6.0 SNP array
- 46. Yale Second cousins in a fully deficient pedigree is a challenging kinship analysis to perform - in 2011 we opted to use an Affymetrix 6.0 SNP array
- 47. Yale
- 48. Yale
- 49. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel • The PCR multiplex must be bigger than scales achieved in forensic MPS before • We also decided to use more informative SNP-based markers: • Tri-allelic SNPs (three possible nucleotide alleles per site = 6 genotypes) • Microhaplotypes in realistically short sequence fragments
- 50. Yale Simulated kinship test LR distributions and linkage adjustments Microhaplotypes The ICMP panel was developed from collaboration between five laboratories and combines a large number of single-site SNPs and microhaplotype loci QIAseq MPS technology Marker selection Optimisation of panel and Qiagen pipeline
- 51. Yale Qiagen first generation MPS technology Andreas Tillmar, Linkoping established the Qiagen 140-SNP ID panel for the MiSeq system, USC adapted it for the Ion PGM
- 52. Yale Andreas Tillmar, Linkoping established the Qiagen 140-SNP ID panel for the MiSeq system, USC adapted it for the Ion PGM Qiagen first generation MPS technology • Good sequence coverage and MPS performance as a third-party kit applicable to both platforms • Qiagen now transitioned to QIAseq chemistry to enable much larger PCR multiplexes and use of multiple primer sets for enhanced sensitivity
- 53. Yale With much larger marker panels applied to kinship tests allowance for linkage becomes important: Linkoping-USC developed 'ILIR' to adjust for linked loci Simulated kinship test LR distributions and linkage adjustments QIAseq MPS technology Rc value estimation
- 54. Yale With much larger marker panels applied to kinship tests allowance for linkage becomes important: Linkoping-USC developed 'ILIR' to adjust for linked loci Simulated kinship test LR distributions and linkage adjustments QIAseq MPS technology Rc value estimation • Not accounting for linkage (here, 20 STRs + 52 SNPs) has a marked effect on LR calculations, particularly when testing related individuals
- 55. Yale Microhaplotypes Marker selection Ken Kidd generously shared microhaplotype data ahead of its publication
- 56. SNP combinations in each haplotype and their phase can only be obtained by sequencing the whole strand - made possible with MPS Yale
- 57. GT, CT, CT 33 = 27 genotype combinations Yale SNP combinations in each haplotype and their phase can only be obtained by sequencing the whole strand - made possible with MPS
- 58. G C C T T T T C C G T T G T C T C T T T C G C T G C T T T C T C T G T C G T T T C C T T T A A C GT, CT, CT 33 = 27 genotype combinations 36 combinations of 8 haplotypes Yale SNP combinations in each haplotype and their phase can only be obtained by sequencing the whole strand - made possible with MPS
- 59. G C C T T T T C C G T T G T C T C T T T C G C T G C T T T C T C T G T C G T T T C C T T T A A C GT, CT, CT 33 = 27 genotype combinations 36 combinations of 8 haplotypes Yale SNP combinations in each haplotype and their phase can only be obtained by sequencing the whole strand - made possible with MPS
- 60. X G C C T T T T C C G T T G T C T C T T T C G C T G C T T T C T C T G T C G T T T C C T T T A A C GT, CT, CT 33 = 27 genotype combinations 36 combinations of 8 haplotypes Yale SNP combinations in each haplotype and their phase can only be obtained by sequencing the whole strand - made possible with MPS
- 61. Yale Microhaplotypes Marker selection Not all microhaplotypes were sufficiently informative - most 2-SNP loci gave tri-allelic patterns with similar power to these loci • Selected 46 from 130 microhaplotypes that have high haplotype Heterozygosity values in most or all 1000 Genomes population groups (i.e. seeking ‘universal’ informativeness) • Reduced the size of some microhaplotypes and traded a reasonable reduction of informativeness for efficiency with degraded DNA
- 62. Yale Microhaplotypes Marker selection Single-site SNP selection was straightforward: compiling a large set of tri-allelic SNPs (using a large catalogue built from 1000 Genomes data)
- 63. Yale Microhaplotypes Marker selection Interestingly, simulations show having many rare minor alleles in tri-allelic SNPs does not reduce kinship likelihoods markedly (in large-scale sets) Simulated kinship test LR distributions and linkage adjustments
- 64. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel
- 65. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel • 1457 markers were incorporated after linkage screening - only 6 sites were eliminated based on primer extension disqualification • 1377 autosomal tri-alleleic SNPs • 34 tri-allelic X chromosome SNPs • 46 microhaplotypes with 2, 3, 4, and 5 SNP combinations
- 66. Why use single nucleotide polymorphisms rather than tried-and-tested STRs for missing persons ID? Forensic MPS as it currently stands and the added advantages of QIAseq chemistry Criteria for building the ICMP missing persons ID panel - moving away from binary markers Characteristics of the markers incorporated into the ICMP missing persons ID panel • 1457 markers were incorporated after linkage screening - only 6 sites were eliminated based on primer extension disqualification • 1377 autosomal tri-alleleic SNPs • 34 tri-allelic X chromosome SNPs • 2832 target enrichment extension primers - 80% of sites with redundant targeting • 46 microhaplotypes with 2, 3, 4, and 5 SNP combinations FP1 FP2
- 67. EUR AFR E ASN S ASN AME 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Typical tri-allelic SNPs for identification - 0.6-0.66 average Heterozygosity EUR AFR E ASN S ASN AME EUR AFR E ASN S ASN AME Yale
- 68. 1411 Tri-allelic SNPs Ranked average Heterozygosity amongst five 1000 Genomes populations (American-East Asian-South Asian-European-African) Yale We were able to combine 1411 tri-allelic SNPs with high levels of average heterozygosity (averaged across five 1000 Genomes population groups) Only 1.5% of tri-allelic SNPs have a lower average Heterozygosity than the 0.5 bi-allelic SNP maximum value (these SNPs had skewed allele frequencies amongst different populations)
- 69. STR sequence variants and Microhaplotypes Yale This microhaplotype exemplifies trading size for informativeness
- 70. STR sequence variants and Microhaplotypes Yale This microhaplotype exemplifies trading size for informativeness
- 71. S ASN EUR AFR Kiddlab microhaplotype sizes ICMP panel microhaplotype sizes 46 Microhaplotypes ranked by descending size - as originally described in Kiddlab list of 130 Average size 128-NT Average size 60.5-NT 16 microhaplotypes had identical sizes, 14 of these were at the extreme size range with an average 55-nucleotide size 65% of Microhaplotypes adopted for the panel had their sizes reduced by an average 67-nucleotides Microhaplotypespaninnucleotides Yale
- 72. MH-D01 39 NT 0.7330 MH-D07 66 NT 0.7434 MH-D50 62 NT 0.6887 MH-D62 59 NT 0.7074 AME E ASN AFR S ASN EUR ACC ACT ATC ATT TCC TCT TTC TTT CAA CAG CTA CTG TAA TAG TTA AAA ACA ACG CAA CCACAA CAT CGT TAA TGA AME E ASN AFR S ASN EUR Typical microhaplotypes for ID - 0.68-0.73 average Heterozygosity Yale
- 73. 46 Microhaplotypes1411 Tri-allelic SNPs Ranked average Heterozygosity amongst five 1000 Genomes populations (American-East Asian-South Asian-European-African) 39% of microhaplotypes have higher average Heterozygosity than 0.66: the tri-allelic SNP maximum value Only 1.5% of tri-allelic SNPs have a lower average Heterozygosity than the 0.5 bi-allelic SNP maximum value (these SNPs had skewed allele frequencies amongst different populations) 98.5% of tri-allelic SNPs and all 46 microhaplotypes are more informative than the best binary SNPs with 0.5-0.5 alleles Yale
- 74. Likelihood ratio distribution comparisons from kinship simulations Unrelated Unrelated vs Second cousinsFirst cousins 0.8 0.6 0.4 0.2 ICMP panel Binary SNPs ICMP panel Binary SNPs Pr(LR>10,1000|HS = 1.000 Pr(LR<0.0001|Un = 1.000 Pr(LR>10,1000|HS = 0.9999 Pr(LR<0.0001|Un = 0.9998 1423 ICMP panel loci 1423 perfect binary SNPs (0.5:0.5) Pr(LR>10,1000|Fsib = 1.000 Pr(LR<0.0001|Un = 1.000 Pr(LR<0.0001|Un = 1.000 0 100 200 300 0 30 60 90 Pr(LR>10,1000|2nd-C = 0.1056 Pr(LR<0.0001|Un = 0.0000 Pr(LR>10,1000|2nd-C = 0.0025 Pr(LR<0.0001|Un = 0.0000 0 4 8 12 Pr(LR>10,1000|1st-C = 0.9885 Pr(LR<0.0001|Un = 0.9341 Pr(LR>10,1000|1st-C = 0.8561 Pr(LR<0.0001|Un = 0.6028 Pr(LR>10,1000|FSib = 1.000 -10 10 20 300 40 log10 LRlog10 LR log10 LRlog10 LR DensityDensity 0.15 0.10 0.05 0.05 0.10 0.10 0.20 Full sibs Unrelated Half sibs ICMP panel Binary SNPs ICMP panel Binary SNPs Unrelated Yale
- 75. Yale • Approximately 7-10% of the tri-allelic sites in ICMP panel are actually segmental duplications giving false three-allele patterns (often in one individual) - so these loci have not been sufficiently well curated by 1000 Genomes at this stage and are now discarded from the multiplex In conclusion: One cautionary observation made during the optimisation of this large panel for routine missing person identification
- 76. Yale In conclusion: One cautionary observation made during the optimisation of this large panel for routine missing person identification • Approximately 7-10% of the tri-allelic sites in ICMP panel are actually segmental duplications giving false three-allele patterns (often in one individual) - so these loci have not been sufficiently well curated by 1000 Genomes at this stage and are now discarded from the multiplex Allelic diversity vs. segmental duplication
- 77. Thanks to everyone at The Forensic Genetics Unit, Santiago