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Implementation of MPS into a Specialist Casework Laboratory
David Ballard
DNA Evidence to Investigative Insights – Berlin, 2016
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Outline
• ForenSeq
• STR Validation
• SNP Validation
• Relationship/Identification
• Specialist Uses
• Mitochondrial DNA Sequencing
• Ancestry Inference
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ForenSeq STRs
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Sequence Variation D2S1338 – Allele 20
20
20
20
20
20
20
TGCC
TTCC
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Sequence Variation D2S1338 – Allele 20
20
20
20
20
20
20
20
20
20
TGCC
TTCC
TCCC
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Sequence Variation D2S1338 – Allele 20
20
20
20
20
20
20
20
20
20
20
20
TGCC
TTCC
TCCC
GTCC
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Sequence-specific population frequencies
0
10
20
30
40
50
60
CE data Caucasian Additional alleles from sequencing Caucasian
CE data Chinese Additional alleles from sequencing Chinese
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Addition of West African frequencies
0
5
10
15
20
25
30
35
40
D16S539 D2S1338 FGA Penta E VWA
Caucasian (n=144)) CE data Caucasian (n=144)) Additional alleles
from sequencing
Chinese (n=169) CE data Chinese (n=169) Additional alleles from
sequencing
West African (n=64) CE data West African (n=64) Additional
alleles from sequencing
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Individual mutations
• No variation observed at D20S482 in 650 alleles- all
simple
[AGAT] repeat unit
• One sample showing single point mutation in one allele
• Not useful in terms of population frequencies
• Could be extremely useful in specific cases
ACAT AGAT AGAT AGAT AGAT AGAT AGAT AGAT AGAT
AGAT AGAT AGAT AGAT AGAT AGAT AGAT
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Sequence Variation D6S1043
AGAT
ACAT
11
11
11
12
12
12
12
12
13
13
13
13
13
14
14
14
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Sequence Variation D6S1043
AGAT
ACAT
11
11
11
12
12
12
12
12
13
13
13
13
13
14
14
14
15
15
15
16
16
17
17
17
17
17
17
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Sequence Variation D6S1043
AGAT
ACAT
18
18
18
18
18
19
19
19
19
19
20
20
20
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Sequence Variation D6S1043
AGAT
ACAT
18
18
18
18
18
19
19
19
19
19
20
20
20
23
23
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Concordance study
• Data generated must align with STR calls generated using
current methodologies
• So far, over 350 samples genotyped, analysed and
compared to CE results from four different STR kits
• 160 White British (3 discordances)
• 164 Chinese (no discordances)
• 60 West African (1 discordance)
• High level of concordance observed
• D22S1045, poorly performing marker: samples appearing
homozygote but heterozygote using the CE-based method
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Primer binding site mutations
Homozygote at D19S433 using the ForenSeq™ and
PowerPlex® kits, and heterozygote using GlobalFiler®
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Flanking region SNPs
• 1 sample had a 7 allele at D7S820, despite it being a 6.3
with all CE-based methods
• Due to rare deletion (rs540346880-0.04% frequency in
Caucasians) in flanking region
CACCAAATATTGGTAATTAAATGTTTACTATAGACTATTTAG
TGAGATTAAAAAAAACTATCAATCTGTCTATCTATCTATCTA
TCTATCTATCTATCGTTAGTTCGTTCTAAACTATGACAAGTG
TTCTATCATACCCTTTAT
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ForenSeq SNPs
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Identity SNP Markers
Autosomal STRs (27)
Y STRs (24)
Identity SNPs (94)
X STRs (7)
Mix A
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Average Read Number per SNP Marker
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Average Heterozygous Balance
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SNP concordance
• One SNP showing a consistent high level of heterozygote
imbalance is rs6955448
• Primer binding site mutation (rs6955464) identified
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Copenhagen SNP Proficiency Test 2015
• ForenSeq SNP calls were compared with those
• SNaPshot
• HID-Ion AmpliSeq Idenitiy Panel (PGM)
• a Qiagen SNP panel ran on the MiSeq
• 6 laboratories participated
• 4 samples
• 367 ForenSeq genotype calls reported - all found
concordant
• SNP rs1528460 failed for 2 samples
• In addition, 7 markers had alleles below the
interpretation
threshold (30 reads) - when checked 3 of these showed allele
dropout.
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Paternity Trios
• We have run 35 confirmed paternity trios to check for
correct SNP inheritence
• 6,580 SNP inheritance events (94 SNPs, 2 parents)
• One discrepancy
• A trio with probable allele dropout in the mother:
• Father AA - 108 reads
• Mother GG – 39 reads
• Child AA – 489 reads
• Re-analysed raw data using our bioinformatics pipeline,
found 9 A reads (19%) in the mother.
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SNP Markers
• Generated allele frequencies
• Checked for Hardy-Weinberg compliance – no significant
deviation for any SNP.
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SNP Pairwise Unrelated Comparisons
• Analysed 5000 pairwise comparisons of unrelated
individuals looking for at least 2 parent-child exclusions
• Theoretically, it is calculated that 2 or more exclusions
will
be observed between unrelated pairs in:
• 99.5 - 99.9% of cases when analysing 80 polymorphic
SNPs
• 99.9 – 100% of cases when analysing 100 polymorphic
SNPs
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Complex relationship testing
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Multiple Mutations
• Three cases analysed with multiple mutations:
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Multiple Mutations
• Three cases analysed with multiple mutations:
• Case 1 – Trio, paternal exclusions in D12S391 & D18S51
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Multiple Mutations
• Three cases analysed with multiple mutations:
• Case 2 – Duo, paternal exclusions in SE33 and D12S391
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Multiple Mutations
• Three cases analysed with multiple mutations:
• Case 3 – Trio, paternal exclusions in D5S818 & CSF.
Maternal or paternal exclusion in D8S1179
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Cousin Relationship
Woman wanting to know if deceased man was her father
Nephews of the man submitted samples
Trying to establish cousin relationship
KitLR For Cousin
LR For Unrelated
NGM/ESI17 2.6
Globalfiler 2.7
44 CE STRs 3.2
+ ForenSeq 57.3
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Cousin Case 2
Trying to establish cousin relationship
LR changed from inconclusive to 17 times more likely to be
cousins
KitLR For Cousin
LR For Unrelated
NGM/ESI17 1.3
Globalfiler 1.6
44 CE STRs 1.8
+ ForenSeq 17.4
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Mitochondrial DNA
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Mitochondrial Hair Analysis
• A mini-mito protocol
• 10 amplicons of 101-190bp
• Improved sensitivity with respect to the CE Sanger
protocol
• Successful, correct, sequencing from PCR products failing
to
display a band when run on a gel
• A TruSeq type protocol employed
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Hair Analysis For Art Authentication
• Two sets of paintings where the authenticity was
questioned
• Hairs extracted from under the paint on the canvas
• Mini-mito protocol run on the MiSeq
• Average coverage of 97,000
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Hair Analysis For Art Authentication
• Case results
• Case 1 – hair from painting didn’t match to the putative
maternal relative
• Case 2 – full match between hair and reference
• EMPOP frequency – 22/26,127
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Ancestry Inference
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King’s Ancestry SNP Panel
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King’s Ancestry SNP Panel
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King’s Ancestry SNP Panel
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Conclusions
• We are approaching a situation where NGS can be applied to
real-life casework for multiple applications
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Acknowledgments
• Laurence Devesse
• Denise Syndercombe Court
• Immy Riethorst
• Federica Giangasparo
• Anastasia Aliferi
• Gabriella Mason-Buck
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DAVID BALLARDDNA ANALYSIS AT KING’SKING’S COLLEGE
LONDONLONDONUK
[email protected]
