The DNASeqEx Project: Exploring STR Sequencing and Global Exchange

Petra Müller 1, Antonio Alonso 2, Pedro A. Barrio 2, Burkhard Berger 1, Martin Bodner 1, Pablo Martin 2, The DNASEQEX Consortium, Walther Parson 1,3

1Institute of Legal Medicine, Medical University of Innsbruck, Austria2National Institute of Toxicology and Forensic Sciences, Madrid Department,

Las Rozas de Madrid, Spain3Forensic Science Program, Penn State University, PA, USA

DNASeqEx - DNA-STR Massive Sequencing & International Information Exchange

EU funded

2 years (2016-2018)

Beneficiaries:The Biology Service of the National Institute of Toxicology and Forensic Science, Madrid, Spain

Antonio Alonso, Pablo Martin, Pedro A. Barrio

Department of Forensic Genetics of the Institute of Legal Medicine and Forensics Science, Berlin, GermanyLutz Roewer, Sascha Willuweit, Steffi Köcher

Institute of Legal Medicine, Medical University of Innsbruck, AustriaWalther Parson, Petra Müller, Burkhard Berger, Martin Bodner

Non-Beneficiary:Institute of Applied Genetics at the University of North Texas Health Science Center, Texas, USA

Bruce Budowle

Promote the implementation of MPS technology for improved STR profiling and international data exchange

→ Inter-laboratory evaluation study

Evaluate the impact of STR sequencing on National DNA databases (EU Prüm, CODIS) → Alonso et al., 2017, FSI Genetics

Facilitate and standardize forensic STR sequence allele nomenclature → NOMAUT - lead Berlin (pending)

Objectives

Consortium Manager

Innsbruck

Bruce Budowle(Consortium Consultant)

Madrid Berlin

Kit Supplier Laboratory

Early Access Applied Biosystems Precision ID Globalfiler Mixture ID™ Panel

TFS Madrid, Innsbruck

Early Access Applied Biosystems Precision ID Globalfiler NGS STR Panel for the Ion S5™

TFSMadrid, Innsbruck

PowerSeq aSTR/mito Kit Promega Innsbruck

PowerSeq 46GY Kit Promega Innsbruck, Berlin

ForenSeq DNA Signature Prep Kit Verogen Innsbruck, Berlin

NGS Prototype Qiagen Innsbruck, Berlin

Evaluated MPS-Kits

Concordance (NIST SRM 2391c A–F)

Reproducibility (GEDNAP persons)

Sensitivity testing (2800M, NIST SRM 2372 A)

♂:♀Mixtures (ratios: 1:1, 1:5, 1:10, 1:15, 1:20)

♂:♂Mixtures (1:1, 1:5, 1:10, 1:15, 1:20)

Extreme Mixtures ♀:♂ (1:100, 1:500, 1:1000)

Mock Casework (GEDNAP stains, GHEP-ISFG)

Population Study (Spain/Austria/Germany)

Addressed Studies

SEQforSTRsAdditional TestingQiagenPromega aSTR/mitoPromega aSTR/Y-STRs

Concordance ConcordanceSensitivity Sensitivity

Reproducibility ReproducibilityMixturesCasework

BerlinMadrid

Innsbruck

Innsbruck (GMI)

Interlocus balanceStutter analysis

Sensitivity

Madrid (INTCF)

EA Applied Biosystems Precision ID Globalfiler Mixture IDTM & Globalfiler

NGS STR Panels for the Ion S5TM

Inter-Laboratory Evaluation Study

General Overview

Early Access Applied Biosystems Precision ID Globalfiler Mixture ID™

113 markers29 aSTRs, 1 Y-STR, 42 SNPs, 2 Y-SNPs, 36 micro-haplotypes, Amelogenin and Y-InDel(rs2032678)

Early Access Applied Biosystems Precision ID Globalfiler NGS STR Panels for the Ion S5™

33 markers29 aSTRs, 1 Y-STR, Amelogenin and Y-InDel(rs2032678)

Interlocus BalanceGlobalfiler Mixture ID™ Panel

Müller et al., manuscriptin preparation

Interlocus balance varies from 4.7 % (D22S1045) to 241.4 % (TH01) com-pared to the calculated expected value (100 %)

GMIINTCF

D2

2S1

04

5D

1S1

67

7A

ME

LY

D1

9S4

33

DY

S 39

1D

3S4

52

9

D1

0S1

24

8F

GA

D4

S24

08

D2

S13

38

D1

8S5

1

D1

2A

TA

63

D1

3S3

17

D1

4S1

43

4D

21

S11

D2

S17

76

D1

2S3

91

vWA

D2

S44

1D

5S2

80

0D

7S8

20

D3

S13

58

CS F

1P

OD

5S8

18

TP

OX

D8

S11

79

D1

S16

56

D6

S47

4D

6S1

04

3A

ME

LXD

16

S53

9T

H0

1

0

5 0

1 5 0

2 0 0

2 5 0

e x p e c te d v a lu e

% h

igh

er

% l

ow

er

Re

lati

ve

ma

rke

r c

ov

era

ge

(%

)

1 0 0

Globalfiler NGS STR Panel™

Müller et al., manuscriptin preparation

Interlocus balance varies from 22.3 % (D22S1045) to 182.7 % (TH01) com-pared to the calculated expected value (100 %)

GMIINTCF

Interlocus Balance

D2

2S1

04

5F

GA

D1

8S5

1D

YS 3

91

AM

ELY

vWA

D1

9S4

33

D4

S24

08

D2

1S1

1D

3S4

52

9D

13

S31

7D

2S1

33

8

D1

0S1

24

8D

7S8

20

D1

2A

TA

63

D5

S28

00

D1

S16

77

D6

S47

4D

5S8

18

D1

4S1

43

4C

S F1

PO

D1

S16

56

D1

2S3

91

D6

S10

43

D2

S44

1D

3S1

35

8T

PO

XD

8S1

17

9D

16

S53

9A

ME

LXT

H0

1

0

5 0

1 5 0

2 0 0

2 5 0

e x p e c te d v a lu e

% h

igh

er

% l

ow

er

Re

lati

ve

ma

rke

r c

ov

era

ge

(%

)

1 0 0

Stutter AnalysisGlobalfiler Mixture ID™ Panel

Müller et al., manuscriptin preparation

GMI

Stutter ratios range from 6.5 % (TH01) to 24.1 % (D22S1045)

Dataset includes single person samples (1 ng DNA input), homo- and heterozygous genotypes as well as isometric allele calls.

T H0 1

T P OX

D5 S 2 8 0 0

D3 S 4 5 2 9

D4 S 2 4 0 8

D2 S 4 4 1

DY S 3 9 1

D7 S 8 2 0

D2 S 1 7 7 6

CS F 1 P O

D6 S 4 7 4

D1 3 S 3 1 7

D5 S 8 1 8

D1 6 S 5 3 9

D6 S 1 0 4 3

D1 4 S 1 4 3 4

FGA

D8 S 1 1 7 9

D1 8 S 5 1

D2 1 S 1 1

D3 S 1 3 5 8

D1 S 1 6 5 6

D2 S 1 3 3 8

D1 S 1 6 7 7

vWA

D1 0 S 1 2 4 8

D1 9 S 4 3 3

D1 2 A

T A6 3

D1 2 S 3 9 1

D2 2 S 1 0 4 5

0

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

stu

tte

r h

eig

ht

(%)

INTCF

Globalfiler NGS STR Panel™

Müller et al., manuscriptin preparation

Stutter ratios range from 5.6 % (TH01) to 18.9 % (D12ATA63)

Dataset includes single person samples (1 ng DNA input), homo- and heterozygous genotypes as well as isometric allele calls.

T H0 1

D4 S 2 4 0 8

D5 S 2 8 0 0

D3 S 4 5 2 9

T P OX

D2 S 4 4 1

D6 S 4 7 4

DY S 3 9 1

D1 3 S 3 1 7

CS F 1 P O

D7 S 8 2 0

D6 S 1 0 4 3

D1 6 S 5 3 9

D8 S 1 1 7 9

D1 4 S 1 4 3 4

D2 1 S 1 1

D1 S 1 6 7 7

D5 S 8 1 8

D3 S 1 3 5 8

D1 0 S 1 2 4 8

vWA

D1 9 S 4 3 3

D2 S 1 3 3 8

D1 S 1 6 5 6

D1 2 S 3 9 1

D1 8 S 5 1

D2 2 S 1 0 4 5

D1 2 A

T A6 3

FGA

0

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

stu

tte

r h

eig

ht

(%)

GMIINTCF

Stutter Analysis

Sensitivity Study24 PCR cycles, NIST 2372A run in duplicate using Globalfiler NGS STR Panel™

Standard PCR conditions display the expected decrease in reads/marker relative to DNA input

Müller et al., manuscriptin preparation

5 0 0 p g 2 5 0 p g 1 2 5 p g 6 2 p g

0

2 0 ,0 0 0

4 0 ,0 0 0

6 0 ,0 0 0

G e n o m ic D N A in p u t / a s s a y (p g )

To

tal

Nu

mb

er

of

Re

ad

s

*

Sensitivity Study28 PCR cycles, NIST 2372A run in duplicate using Globalfiler NGS STR Panel™

Changed cycling conditions affect marker balance

Müller et al., manuscriptin preparation

1 2 5 p g 6 2 p g 3 1 p g 1 5 p g

0

2 0 ,0 0 0

4 0 ,0 0 0

6 0 ,0 0 0

G e n o m ic D N A in p u t / a s s a y (p g )

To

tal

Nu

mb

er

of

Re

ad

s

*

CE result (GF-PCR kit)

D19S433 13.2, 15 Globalfiler Mixture ID™ Globalfiler NGS STR Panel™

GEDNAP 44_S1: Discordance between CE & MPS

14 14 1515

GEDNAP 44_S1

Reference String [Phillips et al., 2018]

CAGGTGGTGTTGGTTACATGAATAAGTTCTTTAGCAGTGATTTCTGATATTTTGGTG

CACCCATTACCCGAATAAAAATCTTCTCTCTTTCTTCCTCTCT[CCTT][CCTT][CCTT]

[CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT]CCTA[CCTT]CTTT[CCTT]CAACAGAATCTTATTCTGTTGCCCAGGCTGGAGTGCAGTGGTACAATTATAG

CTTTTTGCAGCCTCAACCTCCTGGGCTCAAGTGATCTTCCTGCCCCAG

Software Sample Panel MarkerRepeat

StructureReads Sequence String

Sequence-based Data

(MPS)

Length-based

Data (CE)

STRait Razor v2 [King et al., 2017]

44_S1

GF-Mix

D19S433[CCTT]a ccta[CCTT]b cttt

[CCTT]c

529[CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CC

TT]CCTA[CCTT]CTTT[CCTT]CAACAGAATCTTATTCTGTTGCCCAGGCTG14 13.2

140CT[CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT]CCTA[CCTT]CTTT[CCTT]CAACAGAATCTTATTCTGTTGCCCAGGCTG

15 15

GF-STR2139

[CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT]CCTA[CCTT]CTTT[CCTT]CAACAGAATCTTATTCTGTTGCCCAGGCTG

14 13.2

2494CT[CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT][CCTT]CCTA[CCTT]CTTT[CCTT]CAACAGAATCTTATTCTGTTGCCCAGGCTG

15 15

STRait Razor v2s (King et al., 2017).Analytical Threshold: 11 reads (defaul settings).

Müller et al., manuscriptin preparation

Bam files were aligned to GRCh37 (hg 19) using IGV.

2 bp deletion located in the 5’ flanking region of D19S433 (rs745607776).

Müller et al., manuscriptin preparationIntegrative Genomics Viewer (IGV), version 2.3.88

(Robinson et al., 2011 & Thorvaldsdottir et al., 2013)

GEDNAP 44_S1

K.B.Gettings et al., 2016

Conclusion Inter-Laboratory Evaluation Study

Comparable results between INTCF and GMI in terms of: Interlocus balanceStutter ratios

Sensitivity:Fully automated sensitivity testing yielded 98.6 % alleles down to 125 pg (Globalfiler NGS STR Panel™)

Concordance: 367/376 alleles (97.6 %) concordant to known reference (Globalfiler Mixture ID™ Panel)376/376 alleles (100 %) concordant to known reference (Globalfiler NGS STR Panel™)

Additional Remarks

o Fully automated workflow requires low hands-on time in the lab and is easy to handle

o Mixing high and low quality DNA samples in one automated library preparation causes decreased sensitivity

o Software improvements desirable → see alignment according to ISFG recommendations

Innsbruck BerlinMadridApplied Biosystems Precision ID Globalfiler NGS STR for the Ion S5TM (Thermo Fisher Sientific)

Ongoing Task: Population Study

PowerSeq GY46 Kit (Promega) PowerSeq GY46 Kit (Promega)

500 anonymous donors from Spain

250 anonymous donors from Austria

150 anonymous donors from Germany

Alonso et al., 2017, FSI Genetics (published)

Alonso et al., 2018, Electrophoresis (manuscript accepted)

Köcher*, Müller* et al., 2018, FSI Genetics (under review)

Müller et al., 2018, FSI Genetics (in preparation)

Poster Presentations:

Müller et al., ISFG Seoul 2017

Barrio et al., ISFG Seoul 2017

Acknowledgements

Speaker was provided travel and hotel support by Thermo Fisher Scientific for thispresentation, but no remunerationWhen used for purposes other than Human Identification or Paternity Testing theinstruments and software modules cited are for Research Use Only. Not for use indiagnostic procedures.Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promotingany use or application of Thermo Fisher Scientific products presented by third partiesduring this seminar.Information and materials presented or provided by third parties are provided as-is andwithout warranty of any kind, including regarding intellectual property rights and reportedresults.Parties presenting images, text and material represent they have the rights.

Robert LagacéSharon Wootton

Matt PhippsJoseph Chang

Christina Strobl

DNA-STR Massive Sequencing & International Information ExchangeHome/2014/ISFP/AG/LAWX/4000007135

https://www.researchgate.net/project/DNASEQEX