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
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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
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
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
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
dato og ”Enhedens
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
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
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
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
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
454 FILTERED READS - D12S391
ALLELE 1
ERRORS
STUTTERS
ALLELE 2
ERRORS
.
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.
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.
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
rs6736691TGCC 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
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.
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
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
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
SENSITIVITY
• 10 pg – 2 ng
CONCORDANCE
• 10 DANES
• 7 TRACE SAMPLESo 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.
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
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
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
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
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
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
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)
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.
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.
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
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
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
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
ILLUMINA FORENSEQ™ STRs - BETA TEST
ALLELIC BALANCES – PROMISING RESULT
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
ILLUMINA FORENSEQ™ STRs – BETA TEST
DILUTIONS OF DNA – PROMISING RESULTS
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
COMPARISON OF STR KITs
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 + ?
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.
UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE
PGM™ HID SNPs - COVERAGE
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
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
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
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
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
Extraction Kit, Automate Express™ Extraction system, are For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.
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