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

walther.parson@i-med.ac.at

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.