strengths and limitations of ngs for forensic dna analysis · strengths and limitations of ngs for...
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Promega Corporation Promega Corporation
Strengths and Limitations of NGS for Forensic DNA Analysis
Douglas R. Storts, PhD
Head of Research
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NGS versus Capillary Electrophoresis
Advantages Disadvantages
CE
• Established technology • Accepted in court • Relatively easy workflow
• Limited multiplex capability • Complex mixture analysis • Genotyping based on length only
NGS
• Genotyping based on length and sequence • Greater multiplex capability • High dynamic range • Potential improvement to mixture
interpretation • Smaller amplicons (degraded DNA)
• High cost per sample • Large amount of data • Pooling of samples needed to
reduce cost per sample • No guidelines available yet • More complex workflow • Time to result
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PowerPlex® Fusion 6C System
23 autosomal STRs (PI = 2.3 x 10-32), 3 Y-STRs, amel
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PowerPlex® Fusion 6C Allelic Ladder
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PowerSeq™ Systems
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• Amplicon size range is 129-284bp (129-303bp) in the
PowerSeq™ Systems
• Used existing primers where possible to minimize concordance issues
• In contrast, the 27-plex PowerPlex® Fusion 6C System:
Smaller Amplicons
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Extract & Quantify DNA
Targeted Amplification
Library Preparation
Clonal Amplification/Cluster Generation
Sequence
Data Analysis
General Workflow
Illumina MiSeq Reagent kit (MiSeq® Reagent Kit v2)
Variety of reagents
PowerSeq™ Systems
Kit Components • 5X Primer Pair Mix • 5X Master Mix • 2800M Control DNA • Water
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Sample Types
Add reaction mix
Punch FTA® into
plate
FTA® punches
Add reaction mix to
dried punch
Punch card into
plate,
Incubate with
PunchSolution™
NonFTA Punches
Add 2µl of extract
into reaction mix
SwabSolution™
incubation of swab
Swabs
Add up to 15µl of
sample into
reaction
Extract DNA from
sample
Extracted DNA
Cycling time ~85 minutes for all applications
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PowerSeq™ Protocol
Amplification steps are virtually identical to
existing CE-based systems
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Prototype PowerSeq™ Systems
• Current prototype kits
• PowerSeq™ Auto (18 and 24-plex)
• PowerSeq™ Mito (control region)
• PowerSeq™ Y
• PowerSeq™ Auto/Mito
• PowerSeq™ Auto/Y
• PowerSeq™ Auto/Mito/Y
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• SoftGenetics
• Battelle
• STRait Razor
• TSSV
• Others
Data Interpretation
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Recent Manuscript
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Depth of Coverage 500pg Genomic DNA
Zeng, et al. 2015. FSI: Genetics 16:38-47
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Depth of Coverage 62pg Genomic DNA
Zeng, et al. 2015. FSI: Genetics 16:38-47
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PowerSeq™ Auto System Sequence Diversity (18-plex)
Average Stutter-percentage (± StdDev)
Locus n-1_v1 n-1_v2 n+1_v1 n+1_v2 n-2_v1 n-2_v2
CSF1P0 0.063 ± 0.019 ± 0.011 ± 0.007 ± 0.005 ± 0.004 ±
D2S1338 0.088 ± 0.024 0.013 ± 0.006 0.012 ± 0.010 0.010 ± 0.010 0.009 ± 0.004 0.011 ± 0.004
D3S1358 0.091 ± 0.019 ± 0.009 ± 0.008 ± 0.008 ± 0.005 ±
D5S818 0.069 ± 0.017 ± 0.011 ± 0.007 ± 0.006 ± 0.004 ±
D7S820 0.052 ± 0.020 ± 0.008 ± 0.007 ± 0.007 ± 0.005 ±
D8S1179 0.068 ± 0.015 ± 0.009 ± 0.007 ± 0.006 ± 0.004 ±
D13S317 0.056 ± 0.021 ± 0.009 ± 0.007 ± 0.006 ± 0.005 ±
D16S539 0.059 ± 0.021 0.005 ± 0.001 0.009 ± 0.005 ± 0.007 ± 0.008 ±
D18S51 0.090 ± 0.025 ± 0.010 ± 0.009 ± 0.009 ± 0.006 ±
D19S433 0.082 ± 0.026 ± 0.007 ± 0.005 ± 0.008 ± 0.005 ±
D21S11 0.066 ± 0.013 0.012 ± 0.013 0.009 ± 0.004 0.010 ± 0.004 0.008 ± 0.007 0.013 ± 0.004
FGA 0.079 ± 0.021 ± 0.010 ± 0.014 ± 0.009 ± 0.006 ±
PentaD 0.016 ± 0.010 ± 0.008 ± 0.008 ± 0.012 ± 0.037 ±
PentaE 0.038 ± 0.022 ± 0.008 ± 0.009 ± 0.007 ± 0.006 ±
TH01 0.025 ± 0.012 0.004 ± 0.002 0.007 ± 0.007 ± 0.009 ± 0.014 ±
TPOX 0.029 ± 0.013 ± 0.005 ± 0.004 ± 0.006 ± 0.006 ±
vWA 0.080 ± 0.025 0.011 ± 0.010 0.014 ± 0.024 0.005 ± 0.002 0.008 ± 0.006 0.012 ± 0.006
Population Samples Total Alleles
Dutch 102 204
Himalayan 97 194
Pygmee 99 198
Total 298 596 Data courtesy of Peter de Knijff
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PowerSeq™ Auto System Sequence Diversity (18-plex)
0,000
0,200
0,400
0,600
0,800
1,000
Classical STR alleles NGS-SNP alleles NGS-STR alleles
Data courtesy of Peter de Knijff (n=298)
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PowerSeq™ Auto - Expanded STRs to 24plex
Added autosomal STRs to generate multiplex
equivalent to PowerPlex® Fusion
0
10000
20000
30000
40000
50000
60000
70000
80000
Re
ad C
ou
nt
PowerSeq™ Auto Balance
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PowerSeq™ Auto System Sequence Diversity (24-plex)
Data courtesy of the Applied Genetics Group at NIST
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Expanding Beyond Autosomal STRs
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Sequencing of Mitochondrial Control Region
Modification of method described by Eichmann, C. and W. Parson. 2008. 'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples. Int. J. Legal Med, 122:385-388
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PowerSeq™ Mito
0
20000
40000
60000
80000
100000
120000
140000
160000
Alig
ne
d R
ead
s
Start Site of Forward Oligo
Histogram representation of read count data for 2800M DNA library sequenced using MiSeq® v2 chemistry, 1X 260 cycles.
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Combined Auto/Mito Multiplex
0
10000
20000
30000
40000
50000
60000
70000
80000
Alig
ne
d R
ead
s
Balance of Auto/Mito Multiplex
Demonstrate ability to combine STR primers from 18plex with primers for mitochondrial control region into single 5X mix and run in single amplification reaction.
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A Single Multiplex For Multiple Applications
• Single multiplex containing autosomal
loci (23-24), Y STR loci (23) and the
mtDNA control region
• Equivalent to PowerPlex® Fusion,
PowerPlex® Y23 and mitochondrial
control region in a single amplification
PowerSeq™ Mito System
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Aligned reads per locus using 500pg 2800M Control DNA (n=3) To control for sample loading on MiSeq, aligned reads per locus normalized to average of total aligned reads.
0
10000
20000
30000
40000
50000
60000
70000
AM
EL
CSF
1P
O
D1
0S1
24
8
D1
2S3
91
D1
3S3
17
D1
6S5
39
D1
8S5
1
D1
9S4
33
D1
S16
56
D2
1S1
1
D2
2S1
04
5
D2
S13
38
D2
S44
1
D3
S13
58
D5
S81
8
D7
S82
0
D8
S11
79
FGA
PEN
TAD
PEN
TAE
TH0
1
TPO
X
VW
A
DYS
19
DYS
38
5
DYS
38
9I
DYS
38
9II
DYS
39
0
DYS
39
1
DYS
39
2
DYS
39
3
DYS
43
7
DYS
43
8
DYS
43
9
DYS
44
8
DYS
45
6
DYS
45
8
DYS
48
1
DYS
53
3
DYS
54
9
DYS
57
0
DYS
57
6
DYS
63
5
DYS
64
3
GA
TAH
4
Alig
ne
d R
ead
s
PowerSeq™ Auto/Y System
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PowerSeq™ Auto/Mito/Y System
0
10000
20000
30000
40000
50000
60000
AM
EL
CSF
1P
O
D1
0S1
24
8
D1
2S3
91
D1
3S3
17
D1
6S5
39
D1
8S5
1
D1
9S4
33
D1
S16
56
D2
1S1
1
D2
2S1
04
5
D2
S13
38
D2
S44
1
D3
S13
58
D5
S81
8
D7
S82
0
D8
S11
79
DYS
19
DYS
38
5
DYS
38
9I
DYS
38
9II
DYS
39
0
DYS
39
1
DYS
39
2
DYS
39
3
DYS
43
7
DYS
43
8
DYS
43
9
DYS
44
8
DYS
45
6
DYS
45
8
DYS
48
1
DYS
53
3
DYS
54
9
DYS
57
0
DYS
57
6
DYS
63
5
DYS
64
3
FGA
GA
TAH
4
PEN
TAD
PEN
TAE
TH0
1
TPO
X
VW
A
10
9
22
0
31
7
40
2
15
989
16
094
16
197
16
363
16
450
16
533
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Are “We” Ready for Prime Time?
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Relevant Sample Types
• Collaboration with Bruce Budowle
• Manuscript in preparation demonstrating performance of the 24-plex
autosomal STR kit with semen, saliva, blood, and bone samples (single
source and mixtures)
• Data is comparable to CE-based systems
• Able to generate genotypes from 19:1 mixtures
• Able to generate full profiles from 67pg DNA
• Collaboration with Peter de Knijff
• More details to come
• Internal efforts
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PowerPlex® Y23 System
24,000:1 female/male mixture
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0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
AM
EL
CSF
1P
O
D1
0S1
24
8
D1
2S3
91
D1
3S3
17
D1
6S5
39
D1
8S5
1
D1
9S4
33
D1
S16
56
D2
1S1
1
D2
2S1
04
5
D2
S13
38
D2
S44
1
D3
S13
58
D5
S81
8
D7
S82
0
D8
S11
79
FGA
PEN
TAD
PEN
TAE
TH0
1
TPO
X
VW
A
DYS
19
DYS
38
5
DYS
38
9I
DYS
38
9II
DYS
39
0
DYS
39
1
DYS
39
2
DYS
39
3
DYS
43
7
DYS
43
8
DYS
43
9
DYS
44
8
DYS
45
6
DYS
45
8
DYS
48
1
DYS
53
3
DYS
54
9
DYS
57
0
DYS
57
6
DYS
63
5
DYS
64
3
GA
TAH
4
10
9
22
0
31
7
40
2
15
989
16
094
16
197
16
363
16
450
16
533
Tota
l Alig
ne
d R
ead
s
0:1
8:1
24:1
72:1
216:1
PowerSeq™ Auto/Mito/Y System Female:Male Mixtures
Mixtures contained 400ng of female DNA
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PowerSeq™ Auto/Mito/Y System Female:Male Mixtures
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
Tota
l Alig
ned
Rea
ds
8:1
24:1
72:1
216:1
Same graph as before, but only showing the Y-STRs
31 Promega Corporation Promega Corporation
PowerSeq™ Auto/Mito/Y System Female:Male Mixtures
0
20000
40000
60000
80000
100000
120000
140000
AM
EL
CSF
1P
OD
10
S12
48
D1
2S3
91
D1
3S3
17
D1
6S5
39
D1
8S5
1D
19
S43
3
D1
S16
56
D2
1S1
1D
22
S10
45
D2
S13
38
D2
S44
1
D3
S13
58
D5
S81
8D
7S8
20
D8
S11
79
FGA
PEN
TAD
PEN
TAE
TH0
1
TPO
XV
WA
DYS
19
DYS
38
5D
YS3
89
I
DYS
38
9II
DYS
39
0D
YS3
91
DYS
39
2D
YS3
93
DYS
43
7D
YS4
38
DYS
43
9D
YS4
48
DYS
45
6
DYS
45
8D
YS4
81
DYS
53
3
DYS
54
9D
YS5
70
DYS
57
6D
YS6
35
DYS
64
3
GA
TAH
4
Tota
l Alig
ne
d R
ead
s
0:1
8:1
24:1
72:1
216:1
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PowerSeq™ Auto/Mito/Y System Female:Male Mixtures
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
Tota
l Alig
ned
Rea
ds
8:1
24:1
72:1
216:1
Same graph as before, but only showing the Y-STRs
33 Promega Corporation Promega Corporation
PowerSeq™ Auto/Mito/Y System Female:Male Mixtures
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
Tota
l Alig
ned
Rea
ds
800:1
1600:1
3200:1
6400:1
12800:1
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0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
0
1 0 0 0
2 0 0 0
3 0 0 0
P o w e r S e q® A u to /M ito /Y S T R
F e m a le /M a le R a tio T e s t
F e m a le /M a le R a tio
Ali
gn
ed
Re
ad
Co
un
t fo
r Y
ST
Rs
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
P o w e r S e q® A u to /Y S T R
F e m a le /M a le R a tio T e s t
F e m a le /M a le R a tio
Ali
gn
ed
Re
ad
Co
un
t fo
r Y
ST
Rs
0 5 0 0 0 1 0 0 0 0 1 5 0 0 0
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
P o w e r S e q® Y S T R
F e m a le /M a le R a tio T e s t
F e m a le /M a le R a tio
Ali
gn
ed
Re
ad
Co
un
t
Ratio Mean SD
800 4501 2311
1600 3460 1866
3200 1674 728
6400 1043 737
12800 424 256
Ratio Mean SD
8 1297 745
24 623 392
72 219 127
216 95 56
Ratio Mean SD
8 2853 1419
24 1332 692
72 412 224
216 148 89
Summary of Y-STR Data
Real-estate limitation on the MiSeq?
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Future Focus
• Streamline the protocol to be more in alignment with workflow in
a forensic laboratory
• Generate additional data with forensically relevant samples
• Stress test the systems
• Generate additional population data
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Are We Ready for Prime Time?
• Need community consensus on dealing with sequence variants
• Need community consensus on types of markers and regions
interrogated
• Some countries forbid use of ancestral markers
• Is SNP data a burden if you generate an STR profile?
• If you generate the data, you probably need to report the result
• Need streamlined workflow
• Need to reduce costs
• Need more feedback from the community
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Forum for Community Feedback (U.S.)
• Plan to participate in upcoming NIJ grant study (19 month
duration) to define requirements for NGS
• Project coordinated by Battelle with a variety of participating
laboratories (federal, state, and local)
• Armed Forces DNA Identification Laboratory, Dover Air Force Base, DE
• Bureau of Alcohol, Tobacco, Firearms and Explosives, Washington, DC
• California Department of Justice, Sacramento, CA
• Harris County Institute of Forensic Sciences, Houston, TX
• National Institute of Standards and Technology, Gaithersburg, MD
• Pennsylvania State University, State College, PA
• Philadelphia Police Department, Philadelphia, PA
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Acknowledgments
• Promega
• Jaynish Patel
• Spencer Hermanson
• Battelle
• Seth Faith (now at NCSU)
• Esley Heizer, Jr.
• Nancy McMillian
• Richard Guerrieri
• Others
• Leiden Univ. Med. Ctr.
• Peter de Knijff
• Kristiann van der Gaag
• Thirsa Kraaijenbrink
• Rick van Leeuwen
• NIST
• Peter Vallone
• Katherine Gettings
• UNTHSC
• Bruce Budowle
• Xiangpei Zeng
• Jonathan King
• Monika Stoljarova
• David Warshauer
• Bobby LaRue
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Thank you