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