SNP and STR analysis using NGS

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


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


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


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


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


Repeats FlankingPrimer PrimerFlanking

ANALYSIS OF STR NGS DATA

Repeats FlankingFlanking PrimerPrimer

Repeats FlankingFlanking

Repeats FlankingFlanking PrimerPrimer


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


454 FILTERED READS - D12S391

ALLELE 1

ERRORS

STUTTERS

ALLELE 2

ERRORS

.


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.


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.


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


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.


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


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


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.


LT STR 10-PLEX - SGS

ALLELE CALLS, ARTEFACTS AND COVERAGE

STUTTER

ARTIFACT


LT STR 10-PLEX – NOISE

LOSS OR GAIN OF A NUCLEOTIDE IN 47 OF THE 50 MOST FREQUENT ERRORS


LT STRs – ALLELE BALANCES & STUTTERS

COMPLETE CONCONDANCE WITH AMPFLSTR NGM SELECT IN 10 DANES

ALLELE BALANCE STUTTER RATIO


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


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


LT STR 10-PLEX - MIXTURES

MIXTURE 1:1

MIXTURE 1:20

GREEN REPRESENTS THE MINOR COMPONENT


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 resultsPartial Results in all loci

Formalin fixated parafin embedded tissue. Missing person.

EZ1 DNA Investigator (Qiagen) on a BioRobot EZ1 (Qiagen)


Degraded, partialDegraded, partial

Results in all loci

Muscle tissue from severlydegraded body found in appartment (visual ID not possible).

Prepfiler™ Express Forensic DNA Extraction Kit (LT) on Automate Express™ Extraction system (LT)



Results in all loci

Semen from vaginal swab.Rape

Chelex®100 (BioRad) + Amicon®Ultra (Milipore)


FullFull

Full Concordant results

Bone tissue from severlydegraded body recovered from the sea (visual ID not possible).

Phenol/Chloroform 232 AmpFlSTR® IDFL Full Full Concordant results

Muscle tissue from severlydegraded 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


Results in all loci


FORMALIN FIXED PARAFFIN EMBEDDED

PGM HID STR

MPS > PCR-CE


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

rs6736691TGCC

repeatsTTCC

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.


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]




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.


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


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


ILLUMINA FORENSEQ™ STRs - BETA TEST

LOCUS BALANCES – 18 GREEK INDIVIDUALS

PROMISING RESULTS

SEX AUTOSOMAL Y CHROM X CHROM


ILLUMINA FORENSEQ™ STRs - BETA TEST

ALLELIC BALANCES – PROMISING RESULT


ILLUMINA FORENSEQ™ STRs – BETA TEST

DILUTIONS OF DNA – PROMISING RESULTS


COMPARISON OF STR KITs


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


dato og ”Enhedens

SNP SEQUENCING WITH NGS

SNP

59-115bp

Roche GS Junior (500bp)

Illumina MiSeq® (250bp)

Ion Torrent – PGM™System(400bp)

PRIMERPRIMER

READ LENGTH


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


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.


PGM™ HID SNPs - COVERAGE


PGM™ HID SNPs - ALLELE BALANCE

• ALL HETEROZYGOTE ALLELE CALLS FROM THE

CONCORDANCE STUDY

• MINIMUM COVERAGE THRESHOLD: 50-200 READS


• 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


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


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%



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


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+2GAG 3A+1G 2A+2G 1A+3GGG 2A+AG 1A+3G 4G

CORRECTED ACORRECTED MIXTURE PROPORTION = ──────────────

CORRECTED A+G


PGM™ HID SNPs – MIXTURES

ALLELE PROPORTION

THEORETICAL MIXTURE PROPORTION

OB

SE

RV

ED

MIX

TU

RE

PR

OP

OR

TIO

N

THEORETICAL ALLELE BALANCE

0

0

1.0

MISSING DATA

R2 = 0.984


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.


HID-ION AMPLISEQ™ - ANCESTRY PANEL

PROMISING

0

REFERENC SAMPLE 1 ng DNA – 25 PCR CYCLES




BONE SAMPLE – WATER 1 ng DNA – 27 PCR CYCLES


COMPARISON OF SNP KITs - AIMs


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 + DENAMR

EAS

GRL WEST

GRL EAST

OCE

LT


ANCESTRY, TRAITS & SNPs

KIDD

SELDIN

ASIAN

EUROPEAN

AFRICANADMIXED

AMERICAN

FREDERIK

FREDERIK

ILLUMINA


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 withnew technologies

in forensic genetics


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


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.

SNP and STR analysis using NGS

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