SNP and STR analysis using NGS
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"SNP and STR analysis using NGS Niels Morling, MD DMSc Professor of Forensic Genetics Chairman & Director Department of Forensic Medicine Faculty of Health and Medical Sciences University of Copenhagen Denmark"
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SNP and STR analysis using NGS
- 1. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE SNP AND STR ANALYSIS USING NGS Niels Morling, MD DMSc Professor of Forensic Genetics Chairman & Director Department of Forensic Medicine Faculty of Health and Medical Sciences University of Copenhagen Denmark
- 2. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE FORENSIC GENETIC PERSPECTIVES OF NEXT GENERATION SEQUENCING MAY BE USED FOR • STRs FOR ID • SNPs FOR ID • WHOLE mt-GENOME • SNPs FOR PHENOTYPICAL TRAITS • SNPs FOR ANCESTRY • MICROBIAL IDENTIFICATION ALL LOCI SEQUENCED IN ONE INVESTIGATION OTHER USES e.g. • MOLECULAR PATHOLOGY • e.g. GENETIC HEART DISEASES ALSO CALLED • SECOND GENERATION SEQUENCING • MASSIVELY PARALLEL SEQUENCING
- 3. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE NGS IN COPENHAGEN 454 GS JUNIOR SEQUENCING SYSTEM (2009) • LONG STRs MiSeq® (2013) + ForenSeq™ Systems (2014) • FORENSIC SNPs-STRs • mtDNA SEQUENCING • GENES, e.g. HEART • mRNA/miRNA/metDNA ION PGM™ System (2013) -THREE • HID-Ion AmpliSeq™ Identity Panel • HID-Ion AmpliSeq™ Ancestry Panel • LT STR 10-PLEX SEQUENCING
- 4. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE hedens NGS OF STRs – READ LENGTHS Repeat regionFlanking Flanking Primer 70 – 500 bp Roche GS Junior (500 bp) Illumina MiSeq® (250 bp) – PE read Ion Torrent – PGM™ (400 bp) Primer Illumina MiSeq® (250 bp) – PE read
- 5. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE Fordyce et al. Biotechniques 2011; 51: 127-133 • AMPLICON SEQUENCING WITH MULTIPLEX IDENTIFIERS (MIDs) IN THE PCR PRIMER SEQUENCE • QUANTIFY AND POOL AMPLICONS INTO A LIBRARY • emPCR AND SEQUENCING 454 STR SEQUENCING WITH GS JUNIOR
- 6. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE Repeats FlankingPrimer PrimerFlanking ANALYSIS OF STR NGS DATA Repeats FlankingFlanking PrimerPrimer Repeats FlankingFlanking Repeats FlankingFlanking PrimerPrimer
- 7. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE Repeats FlankingFlanking • FILTER BY SEQUENCES OF PRIMER OR FLANKING REGIONS THE PRESENCE OF AT LEAST ONE PRIMER BINDING REGION IS PREFERABLE • SORT BY PRIMER/FLANKING SEQUENCES BOTH ENDS MUST BE PRESENT • TRIM THE READS KEEP THE FLANKING REGIONS • GENERATE A TABLE WITH STR SEQUENCES AND FRAGMENT LENGTHS • GENERATE A TABLE WITH SEQUENCES, FRAGMENT LENGTHS AND NOs OF READS OF EACH LENGTHS FORDYCE ET AL 2011 ANALYSIS OF STR NGS DATA
- 8. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE 454 FILTERED READS - D12S391 ALLELE 1 ERRORS STUTTERS ALLELE 2 ERRORS .
- 9. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE 454 - SEQUENCES OF CSF1PO ALLELES Fordyce et al. High-throughput sequencing of core STR loci used for forensic genetic investigations using the Roche Genome Sequencer FLX platform. Biotechniques 2011; 51: 127-33.
- 10. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE 454 - SEQUENCES OF D21S11 ALLELES PATERNITY CASE Rockenbauer et al. Sequences of microvariant/‘‘off-ladder’’ STR alleles. Forensic Science International Genetics Supplement Series 2011; 3: e204-5.
- 11. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE rs6736691 TGCC repeats TTCC repeats rs9678338 Rockenbauer et al. FSI Genet 2014; 8: 68-72 Alsgaard et al. FSI Genet Suppl Serie 2013; 4: e218-e219 454 - SEQUENCES OF D2S1338 ALLELES EVALUATION OF MUTATIONS IN • D21S11, D12S391, D2S1338, D3S1358 • CONFIRMED THAT SINGLESTEP MUTATIONS ARE THE MOST FREQUENT ONES
- 12. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LESSONS FROM 454 SEQUENCING OF D21S11, D12S391 AND D3S1358 DATABASE WITH 394 ALLELES IN 197 UNRELATED DANES Number of alleles PCR-CE SGS D21S11 13 29 D12S391 15 53 D3S1358 8 17 Total 36 99 Forensic statistics PCR-CE SGS Power of discrimination 0.9999 0.999995 Paternity exclusion power 97.1 99.2 PI 59.2 415.0 mPI 16.1 82.4 • APPROXIMATELY 30% OF THE ONE-ALLELE TYPES OBTAINED WITH PCR-CE TURNED OUT TO BE FROM HETEROZYGOUS INDIVIDUALS • ALLLELE 21 OF D12S391: 8 MICRO-VARIANTS • COMPLEX STRs ARE ESPECIALLY USEFUL FOR MIXTURE INTERPRETATION Typical PI Gelardi et al. Second generation sequencing of three STRs D3S1358, D12S391 and D21S11 in Danes and a new nomenclature for sequenced STR alleles. Forensic Sci Int Genet 2014; 12: 38-41.
- 13. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LIFE TECHNOLOGIES (LT) STR 10-PLEX A 10-PLEX DESIGNED USING AMPLISEQ™ Kit SYSTEMS • AMELOGENIN • CSF1PO • D16S539 • D3S1358 • D5S818 • D7S820 • D8S1179 • TH01 • TPOX • VWA • AFTER PCR, PRIMERS ARE DIGESTED • AMPLICONS 75 – 170 BP • SEQUENCE WITH 314V2 CHIP OR OTHER ON PGM • HID STR GENOTYPER PLUGIN FOR ANALYSIS
- 14. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ADJUSTING STR PRIMERS FOR NGS OWN 454 GS JUNIOR WORK & PGM HID STR SGS REDUCE IMBALANCE BETWEEN STR SYSTEMS BY • AMPLICON LENGTHS WITHIN 150 BP AND/OR • +/-20% 20% RANGE
- 15. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE SENSITIVITY • 10 pg – 2 ng CONCORDANCE • 10 DANES • 7 TRACE SAMPLES o RED HOODIE FROM ROBBERY o MUSCLE TISSUE FROM DECOMPOSED BODY (ID CASE) o FFPE TISSUE (ID CASE) o VIGINAL SWAB FROM RAPE CASE o BONE FROM BODY WASHED UP ON A BEACH (ID CASE) o MUSCLE TISSUE FROM BODY WASHED UP ON A BEACH (ID CASE) o FFPE TISSUE (ID CASE) • COMPARISON WITH ISO17025 ACCREDITED STR TYPING AMPFlSTR NGMSELECT OR IDENTIFILER MIXTURES (MALE/FEMALE) • 1:1000, 1:100, 1:10, 1:5, 1:2, 1:1 LT STR 10-PLEX – PLAN Fordyce et al. Forensic Sci Int Genet 2015; 14: 132-40.
- 16. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STR 10-PLEX - SGS ALLELE CALLS, ARTEFACTS AND COVERAGE STUTTER ARTIFACT
- 17. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STR 10-PLEX – NOISE LOSS OR GAIN OF A NUCLEOTIDE IN 47 OF THE 50 MOST FREQUENT ERRORS
- 18. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STRs – ALLELE BALANCES & STUTTERS COMPLETE CONCONDANCE WITH AMPFLSTR NGM SELECT IN 10 DANES ALLELE BALANCE STUTTER RATIO
- 19. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STRs - ALLELE AND LOCUS DROP-OUT COVERAGE WITH CORRECT SEQUENCE 1 10 100 1 10 100 DNA (ng) N ALLELE DROP-OUT (%) LOCUS DROP-OUT (%) 2.00 40 0 0 0 0 0 0 1.00 40 0 0 0 0 0 0 0.50 40 0 0 0 0 0 0 0.20 40 0 0 0 0 0 0 0.10 80 0 0 0 0 0 0.01 0.05 80 0 0 0 0 0 0 0.02 40 0.05 0.13 0.13 0 0 0 0.01 40 0.05 0.20 0.23 0.05 0.05 0.05 2 INDIVIDUALS 2 - 4 LIBRARIES
- 20. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STR 10-PLEX vs STR TYPING BY PCR-CE COMPLETE CONCONDANCE BETWEEN (1) NGS AND (2) PCR-CE DATA WITH AmpFLSTR® NGM SELECT IN 10 DANES
- 21. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STR 10-PLEX - MIXTURES MIXTURE 1:1 MIXTURE 1:20 GREEN REPRESENTS THE MINOR COMPONENT
- 22. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE LT STR 10-PLEX - CHALLENGING MATERIAL Sample description Extraction method DNA (ng/ul) Kit CE Results NGS Results Red hoodie from a robbery. Sample taken from sleeve close to wrist. Prepfiler™ Express Forensic DNA Extraction Kit (LT) on Automate Express™ Extraction system (LT) 2.3 AmpFlSTR® IDFL Plus AmpFlSTR®NGMSelect ™ No results Partial Results in all loci Formalin fixated parafin embedded tissue. Missing person. EZ1 DNA Investigator (Qiagen) on a BioRobot EZ1 (Qiagen) 0.597 AmpFlSTR® IDFL Plus AmpFlSTR®NGMSelect ™ Degraded, partial Degraded, partial Results in all loci Muscle tissue from severly degraded body found in appartment (visual ID not possible). Prepfiler™ Express Forensic DNA Extraction Kit (LT) on Automate Express™ Extraction system (LT) 48.9 AmpFlSTR® IDFL Plus AmpFlSTR®NGMSelect ™ Degraded, partial Degraded, partial Results in all loci Semen from vaginal swab. Rape Chelex®100 (BioRad) + Amicon®Ultra (Milipore) 0.342 AmpFlSTR® IDFL Plus AmpFlSTR®NGMSelect ™ Full Full Full Concordant results Bone tissue from severly degraded body recovered from the sea (visual ID not possible). Phenol/Chloroform 232 AmpFlSTR® IDFL Full Full Concordant results Muscle tissue from severly degraded body recovered from the sea (visual ID not possible). Phenol/Chloroform 23 AmpFlSTR® IDFL Full Full Concordant results Formalin fixated parrafin embedded tissue. Missing person. Phenol/Chloroform 1.88 AmpFlSTR® IDFL AmpFlSTR® SEfilerPlus Degraded, partial Degraded, partial Results in all loci
- 23. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE FORMALIN FIXED PARAFFIN EMBEDDED PGM HID STR MPS > PCR-CE
- 24. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE NOMENCLATURE OF STR SEQUENCES • LOCUS NAME USED IN FORENSIC GENETICS • LENGTH OF STR • SEQUENCES AND NUMBERS OF REPEAT UNITS • SEQUENCE DETAILS • POLYMORPHISMS IN THE FLANKING REGION (SNPs AND INDELs) ALLELE 1: D2S1338[18]TGCC[6]TTCC[12]rs6736691T ALLELE 2: D2S1338[23]TGCC[7]TTCC[13]GTCC[1]TTCC[2]rs6736691G rs6736691 TGCC repeats TTCC repeats rs9678338 The Human Genome Variation Society - http://www.hgvs.org STRbase - http://www.cstl.nist.gov/div831/strbase Gelardi et al. Second generation sequencing of three STRs D3S1358, D12S391 and D21S11 in Danes and a new nomenclature for sequenced STR alleles. Forensic Sci Int Genet 2014; 12: 38-41.
- 25. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE SYSTEM ALLELE SEQUENCE TPOX 10 AATG[10] TPOX 11 AATG[11] CSF1PO 11 TATC[11] CSF1PO 12 TATC[12] D5S818 11 ATCT[11]rs73801920[A]rs25768[G] D5S818 11 ATCT[11]rs73801920[C]rs25768[G] D7S820 11 TATC[11]rs16887642[G] D16S539 11 GATA[11]rs11642858[A] D16S539 13 GATA[13]rs11642858[A] D3S1358 15 TATC[2]TGTC[2]TATC[11] D3S1358 18 TATC[2]TGTC[2]TATC[14] D8S1179 15 TATC[3]TGTC[1]TATC[11] D8S1179 13 TATC[2]TGTC[1]TATC[10] vWA 17 TAGA[12]CAGA[4]TAGA[1]rs75219269[A] vWA 18 TAGA[13]CAGA[4]TAGA[1]rs75219269[A] TH01 9.3 AATG[6]ATG[1]AATG[3] TH01 9 AATG[9] LT STR 10-PLEX – STRs OF AN INDIVIDUAL Gelardi et al. Forensic Sci Int Genet 2014; 12: 38-41.
- 26. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE SUMMARY OF 454, LT & ILLUMINA WORK NGS WORKS WELL FOR SNPs, STRs AND MITOCHONDRIAL DNA • GENOTYPING √ • SENSITIVITY √ • ALLELE BALANCE √ • REPRODUCIBILITY √ • TRACE SAMPLES √ • MIXTURES √ • DATA ANALYSIS NEEDS IMPROVEMENTS • SIMPLER, FASTER, LESS HANDS-ON – AUTOMATION OF • LABORATORY PROCEDURES • DATA ANALYSIS • INTERNATIONAL NOMENCLATURE MUST BE IN PLACE SOON
- 27. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ILLUMINA FORENSEQ™ SNP-STRs BETA TEST OF ‘MIX B’ – 235 GENETIC MARKERS STRs • AUTOSOMAL 28 • Y 25 • X 9 SNPs • AUTOSOMAL 95 • PHENOTYPIC 24 • ANCESTRY 54 COPENHAGEN PART OF A COLLABORATIVE EXERCISE – 13 LABS DATA ANALYSED BY COPENHAGEN AND ILLUMINA
- 28. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ILLUMINA FORENSEQ™ STRs - BETA TEST LOCUS BALANCES – 18 GREEK INDIVIDUALS PROMISING RESULTS SEX AUTOSOMAL Y CHROM X CHROM
- 29. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ILLUMINA FORENSEQ™ STRs - BETA TEST ALLELIC BALANCES – PROMISING RESULT
- 30. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ILLUMINA FORENSEQ™ STRs – BETA TEST DILUTIONS OF DNA – PROMISING RESULTS
- 31. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE COMPARISON OF STR KITs
- 32. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE FUTURE DIRECTIONS OF STRs WITH NGS • COMMON STR LOCI – ESPECIALLY SHORT, POLYMORPHIC LOCI • NEW PRIMERS CLOSER TO STR REPEATS • ADVANTAGE FOR BALANCING MULTIPLEXES • ADVANTAGE FOR DEGRADED SAMPLES • IMPROVEMENT OF SEQUENCING CHEMISTRIES, etc. • LONGER READ LENGTHS FOR STRs • LARGER OUTPUT • INTEGRATED LABORATORY AUTOMATION • CRIME CASES: SUPPLEMENT WITH SNPs FOR HID + ANCESTRY + PHENOTYPICAL TRAITS +? • RELATIONSHIP TESTING: STRs + SNPs FOR IDENTIFICATION + ? • DEDICATED FORENSIC GENETIC INTERPRETATION SOFTWARE TOOLS
- 33. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE hedens SNP SEQUENCING WITH NGS SNP 59-115bp Roche GS Junior (500bp) Illumina MiSeq® (250bp) Ion Torrent – PGM™System (400bp) PRIMERPRIMER READ LENGTH
- 34. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNP ASSAY Predecessor of HID-Ion AmpliSeq™ Identity Panel HID SNP PANEL V2.2: • 51 SNPFORID SNPS* • 89 II SNPS† • 33 Y-CHROMOSOME MARKERS *Sanchez et al. Electrophoresis 2006; 27: 1713-24 †Pakstis et al. Hum Genet 2010; 127: 315-24 HID SNP PANEL V2.3: • 48 SNPFORID SNPS* • 43 II SNPS† • 30 Y-CHROMOSOME MARKERS AUTOSOMAL SNPS WERE SELECTED FOR HUMAN IDENTIFICATION PURPOSES*† MATCH PROBABILITIES IN THE RANGE OF 10-18 FOR EACH PANEL Y-CHROMOSOME MARKERS IDENTIFY THE MAJOR Y-HAPLOGROUPS
- 35. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNP - PROJECT LT AMPLISEQ™ HID SNP PANEL VERSION 2.2 & Ion PGM™ System 136 AUTOSOMAL SNPs 33 Y-CHROMOSOME SNPs 9 SNPs EXCLUDED DUE TO POOR PERFORMANCE rs2399332, rs1029047, rs1358856, rs10776839, rs4530059, rs8037429, rs430046, rs1031825, rs1523537 160 SNPs ANALYSED 2.5 ng DNA FROM ONE INDIVIDUAL 4 LIBRARIES -> POOLED 2 SEQUENCES OF EACH LIBRARY POOL ON 314 CHIPs Børsting et al. Evaluation of the Ion Torrent™ HID SNP 169-plex: A SNP typing assay developed for human identification by second generation sequencing. Forensic Sci Int Genet 2014; 12: 144-54.
- 36. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNPs - COVERAGE
- 37. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNPs - ALLELE BALANCE • ALL HETEROZYGOTE ALLELE CALLS FROM THE CONCORDANCE STUDY • MINIMUM COVERAGE THRESHOLD: 50-200 READS
- 38. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE • CONCORDANCE WITH SNPforID ASSAY EXCEPT FOR TWO SNPS • EXPECTED Y-HAPLOGROUP DESIGNATIONS WERE OBTAINED FOR ALL SAMPLES • EIGHT SNPS WITH LARGE VARIATIONS IN ALLELE BALANCES • SIX SNPS HAD SEQUENCING BIAS IN EITHER FORWARD OR REVERSE DIRECTION • TWELVE SNPS FAILED THE HW-TEST • VERY FEW (TYPICALLY < 5) READS WITH BASE CALLS THAT DIFFERED FROM THE GENOTYPE CALL PGM™ - CONCORDANCE WITH SNPforID
- 39. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNPs - SENSITIVITY • 100 pg – 10 ng DNA • 44 IRAQI MALES TYPED TWICE • COMPARISON WITH • ACCREDITATED PCR-CE SNPforID ASSAY* • PREVIOUSLY IDENTIFIED Y-HAPLOGROUPS† *Børsting et al., FSI Genet 2009; 4: 34-42 †Sanchez et al., Eur J Human Genet 2005; 13: 856-66
- 40. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNPs - SENSITIVITY SNPs DNA (ng/μl) Number of locus dropouts Number of allele dropouts Correctly typed SNPs* Autosomal 0.1 256 7 48.2% Autosomal 0.2 73 8 83.9% Autosomal 0.5 3 1 99.2% Autosomal 1 3 0 99.4% Autosomal 2 0 0 100% Autosomal 5 0 0 100% Autosomal 10 0 0 100% Y 0.1 33 - 48.4% Y 0.2 30 - 53.1% Y 0.5 11 - 82.8% Y 1 5 - 92.2% Y 2 0 - 100% Y 5 0 - 100% Y 10 0 - 100% • ONLY ALLELE DROP-OUT WHEN COVERAGE < 50 READS • NO ALLELE DROP-IN
- 41. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNPs - MIXTURE DETECTION • AMPLISEQ HID SNP PANEL VERSION 2.2 & LT IONTORRENT PGM • MIXTURES OF DNA FROM TWO INDIVIDUALS WITH KNOWN SNP TYPES • MIXTURE PROPORTIONS: 1,000:1, 100:1, 10:1, 5:1, 2:1, 1:1, 1:2, ... • AMOUNT OF DNA 10 pg – 10 ng • PARTS OF THE DATA MISSING • DATA OF 9 POORLY PERFORMING SNPs EXCLUDED • NUCLEOTIDE READS < 10 EXCLUDED • GENOTYPE COMBINATIONS TAKEN INTO ACCOUNT AA AG GG ────────────────────── AA 4A 3A+1G 2A+2G AG 3A+1G 2A+2G 1A+3G GG 2A+AG 1A+3G 4G CORRECTED A CORRECTED MIXTURE PROPORTION = ────────────── CORRECTED A+G
- 42. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE PGM™ HID SNPs – MIXTURES ALLELE PROPORTION THEORETICAL MIXTURE PROPORTION OBSERVEDMIXTUREPROPORTION THEORETICAL ALLELE BALANCE 0 0 1.0 MISSING DATA R2 = 0.984
- 43. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE HID-ION AMPLISEQ™ - LATEST VERSIONS HID-Ion AmpliSeq™ Ancestry Panel • 165 SNPs HID-Ion AmpliSeq™ Identity Panel • 90 AUTOSOMAL SNPs • 30 Y CHROMOSOMAL SNPs FIRST EXPERIMENTS STARTED IN COPENHAGEN • TRIMMING FOR CRIME AND ID CASES • DNA AMOUNT • PCR CYCLE IN FIRST PCR • ETC.
- 44. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE HID-ION AMPLISEQ™ - ANCESTRY PANEL PROMISING 0 REFERENC SAMPLE 1 ng DNA – 25 PCR CYCLES REFERENC SAMPLE 1 ng DNA – 27 PCR CYCLES REFERENC SAMPLE 1 ng DNA – 29 PCR CYCLES REFERENC SAMPLE 1 ng DNA – 30 PCR CYCLES BONE SAMPLE – WATER 1 ng DNA – 27 PCR CYCLES
- 45. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE COMPARISON OF SNP KITs - AIMs
- 46. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE KIDD SNPs SELDIN SNPs UI1 UI3 Y-hg H mtDNA-hg R Y-hg E1b1b mtDNA-hg L3f1b ANCESTRY BY SNP TYPING OF AIMs EUROFORGEN SNPs AFR SOM EUR + DEN AMR EAS GRL WEST GRL EAST OCE LT
- 47. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ANCESTRY, TRAITS & SNPs KIDD SELDIN ASIAN EUROPEAN AFRICAN ADMIXED AMERICAN FREDERIK FREDERIK ILLUMINA
- 48. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE NEW DNA SEQUENCING METHODS IN FORENSIC GENETICS CRITICAL TESTING OF • RELIABILITY • SENSITIVITY • SPECIFICITY • RISK OF CONTAMINATION • RESOLUTION OF DNA MIXTURES • WEIGHT OF THE EVIDENCE • etc. Be careful with new technologies in forensic genetics
- 49. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE THE COPENHAGEN NGS GROUP Jeppe Dyrberg Andersen, MSc, PhD Student Anders Buchard, MSc, PhD, Forensic Geneticist Claus Børsting, MSc, PhD, Forensic Senior Advisor Sofie Christiansen, MSc, PhD Student Sarah Louise Fordyce, MSc, PhD, Postdoc Susanne Friis, MSc, Forensic Geneticist Christin Løth Hertz, MD, PhD Student Marie-Louise Kampmann, MSc, PhD, Postdoc Carmen Tomas Mas, MSc, PhD, Forensic Geneticist Helle Smidt Mogensen, MSc, PhD, Forensic Geneticist Niels Morling, MD, DMSc, Professor Jill Olofsson, MSc, PhD Student Vania Pereira, MSc, PhD, Postdoc Eszter Rockenbauer, MSc, PhD, Forensic Geneticist Goncalo Themudo, MSc, PhD, Postdoc
- 50. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
- 51. UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE ION TORRENT PGMTM, HID-Ion AmpliSeq™ Identity Panel, HID- Ion AmpliSeq™ Ancestry Panel, AmpFlSTR® IDFL Plus, AmpFlSTR®NGMSelect™, AmpFlSTR® IDFL, AmpFlSTR® SEfilerPlus, Prepfiler™ Express Forensic DNA Extraction Kit, Automate Express™ Extraction system, are For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. Life Technologies and its affiliates are not endorsing, recommending, or promoting any use or application of Life Technologies products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so. Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration.