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Massively parallel sequencing in forensic genetics
Human Identification User Meeting Madrid, Mar 03 2015
Dr. Walther Parson
assoc. Prof. Institute of Legal Medicine, Innsbruck, Austria
adj. Prof. Penn State University, PA, USA
DNA Degradation by DNase treatment
DNA Degradation by DNase treatment
mtDNA quants aDNA
1
10
100
1000
10000
100000
51bp 57bp 143bp
log
10 m
tDN
A [
GE
/µl]
qPCR target size
aDNA from medieval remains (5th/6th c. AD)
FA10004
FA10014
mtGenome MPS of midi size amplicons
MPS strategy for highly degraded DNA
MtDNA control region PEC - FA10014
1 8300 16569
PCR: 14 CR primers
Library: gDNA Fragment Library Kit
Sequencing: PGM 314 V2, OT2, 200 bp Sequencing
CV around 15-150
MtDNA control region PEC - FA10014
Sanger (2012) 60 mtGE/µl [143bp]
16126C 16163G 16186T 16189C 16294T 16355T16519C
73G 152C
PEC (2015) 20.000 mtGE/µl [57bp]16126C 16163G 16186T 16189C 16294T 16355T16519C 73G 152C 263G 315.1C 709A 750G15884A15928A
hg T1a12
Phylotree, b.16
MtDNA control region PEC - FA10008
Sanger (2012) 6,328 mtGE/µl [143bp]
73G 263G
315.1C523DEL524DEL
PEC (2015) n.a. [60bp]
73G 263G
315.1C523DEL524DEL
750G8269A14365T
hg H4a1a1a
Phylotree, b.16
MtDNA control region PEC - FA11014
Sanger (2012) 0 mtGE/µl [143bp]
no result
PEC (2015) 7 mtGE/µl [51bp], 2 mtGE/µl [57bp]152C 263G 315.1C 750G14155T14869C16519C
hg H16d
Phylotree, b.16
Full match in overlapping range with GenBank® sequences
AY495127.2, JN037470.1, JN247624.1 (all H16d)
NTC clean
mtDNA quants aDNA
0
50
100
150
200
250
300
350
400
450
51bp 57bp 143bp
mtD
NA
[G
E/µ
l]
qPCR target size
Tooth Urnfield Culture (c.1300-750 BC)
S llfried4
Sample donated by M. Teschler-Nicola, NHM Vienna
0
5
10
15
20
25
30
35
40
45
1602
4
1605
1
1607
8
1610
5
1613
2
1615
9
1618
6
1621
3
1624
0
1626
7
1629
4
1632
1
1634
8
1637
5
1640
2
1642
9
1645
6
1648
2
1650
9
1653
6
1656
3 21 48 77 104
131
158
185
212
239
269
297
322
349
378
405
433
460
490
519
553
Num
ber o
f Obs
erva
tions
16093
16519
146
152
1618316189 195
204
16192215
PHP in 5,015 Control Regions
Irwin et al (2009)
6%6% - CR heteroplasmy rate (max. 3/Ind)
Significant correlation (p < 0.001) between heteroplasmy hotspots and substitution hotspots
PHP in 588 mtGenomes
24%24% - CodR heteroplasmy rate (max 3/ind)
Random distribution across mtGenome
1000 Genomes Project, mean coverage of ∼2,000x
∼90% of the individuals carry at least one heteroplasmy (1% MAF)
Positive correlation between substitution rates and heteroplasmy
rates
Inspection of Dataset (Dataset S1):
15 samples with 20 or more PHPs (up to 71!)
80.7% of the 584 PHPs occurred at hg-specific sites
HG00740: 90% of the 71 PHPs can be ascribed to either hg L1b1a7a
and B2b3a
HG01108: 50 and 12 of the 69 PHPs are diagnostic for hgs L0a1a2
and M7c1b
No hg-specific PHPs remain when a 15% MAF is applied for
HG00740 and a 25% MAF for HG01108
Literature Review mtGenome PHP and NGS
0
10
20
30
40
50
60
70
80
90
100
0,0%
10,0%
20,0%
30,0%
40,0%
50,0%
60,0%
70,0%
80,0%
90,0%
100,0%
Li 2010 Sosa 2012 Ramos 2013 Diroma 2014 King 2014 Ye 2014, revised
Rebolledo-Jaramillo 2014
Just 2015 Skonieczna
2015
Ma
xim
um
# o
f P
HP f
or
a s
ing
le i
nd
ivid
ua
l
% o
f In
div
idu
als
/lin
ea
ge
s w
ith P
HP
10-20% 2% 1% <1% Detec on Threshold/MAF:
Just et al, under review
Maximum PHP in a single individual % of coding region PHP observed only once
Summary
PEC
PEC assay results in concordant mtDNA sequences
PEC assay (also) for shorter DNA fragments
PEC assay seems more sensitive
Problem of interpreting contamination
PHP
Careful phylogenetic interpretation of PHP
Reliable studies suggest random distribution of PHP in codR
Acknowledgements
FP7-SEC-2011-285487
Translational Research project L397
“EMPOP–an innovative human mtDNA database”
2011-MU-MU-K402
Maximizing mtDNA Testing Potential with the Generation
of High-Quality mtGenome Reference Data
Quants - Cordula Berger, Harald Niederstätter
PEC - Mayra Eduardoff
PGM - Mayra, Catarina Xavier
Research project P22880-B12
“Genetic discovery of an early medieval Alpine population ”
Ion PGMTM System is For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.
Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration.