Next Generation Sequencing Activities at NIST · 2020. 5. 5. · •HID-Ion Ampliseq Identity Panel (IISNP) –120 markers in a single PCR reaction –Amplified regions 33 bp to 192

Next Generation Sequencing Activities at NIST

NGS Workshop Mid-Atlantic Association of Forensic Science

May 19, 2014

Katherine Butler Gettings, Ph.D. Research Biologist, Applied Genetics Group

National Institute of Standards and Technology

I will mention commercial STR kit names and information, but I am in no way attempting to endorse any specific products.

NIST Disclaimer: Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.

Points of view are mine and do not necessarily represent the official position of the National Institute of Standards and Technology or the U.S. Department of Justice. Our group receives or

has received funding from the FBI Laboratory and the National Institute of Justice.

Disclaimer

Mid-Atlantic Association of Forensic Science Next Generation Sequencing Workshop

May 19, 2014

Outline

Background

NGS of Forensic DNA markers

– STRs

– mtDNA

– Single Nucleotide Polymorphisms (SNPs)

NGS on the PGM- Ampliseq workflow

Experimental data

– HID-Ion Ampliseq Identity Panel

– HID-Ion Ampliseq Ancestry Panel

What’s in a name???

Next-generation sequencing

Massively parallel sequencing

Second-generation sequencing

Third-generation sequencing

NGS

High-throughput sequencing

Next-generation genomics

Whole-genome sequencing

Parallel Sequencing ‘A million capillary Sanger sequencer’

Parallel Sequencing

‘A million capillary Sanger sequencer’

• Clonal vs population amplification

• Shorter reads (Range 75 to 400)

• Errors are more ‘detectable’

• High coverage 100 – 1000 - 10,000x

• Rely more on informatics to assemble millions of short reads

http://electronicsbyexamples.blogspot.com/2013/03/milestones-in-digital-electronics.html

http://blog.trentonsystems.com/moores-law-pushing-processor-technology-to-14-nanometers/

Forensic NGS Applications

• Short Tandem Repeats (STRs)

– PCR fragment-length polymorphisms

• Mitochondrial DNA (mtDNA)

– Sanger sequencing

• Single Nucleotide Polymorphisms (SNPs)

Capillary electrophoresis electropherogram

NGS of Forensic STR Loci


D8S1179

D21S11

D7S820

CSF1PO

D3S1358

TH01

D13S317

D16S539

D2S1338

D19S433

vWA

TPOX

D18S51

A

D5S818

FGA

Sizes of largest observed alleles (not including primer binding/flanking region)


D8S1179

D21S11

D7S820

CSF1PO

D3S1358

TH01

D13S317

D16S539

vWA

TPOX

D18S51

A

D5S818

FGA

Sizes of largest observed alleles (not including primer binding/flanking region) D2S1338

D19S433


D21S11: Individual appears homozygous by CE but different sequencing composition shown with NGS.

D21S11: Individuals appear homozygous by CE but different sequencing composition shown with NGS.



STR sequence data from 2391c, Component A:

• Truseq Library Prep

• MiSeq sequencing

• STRait Razor data parsing

• R script (NIST) data viewer

Forensic DNA Markers






http://www.orchidcellmark.ca http://remf.dartmouth.edu/images/mammalianLungTEM/source/8.html

Mitochondria

www.wikipedia.org

Maternally inherited Circular genome

• Increase in variants by whole genome analysis

(based on analysis of 3 SRM samples)

mtDNA Information

Over ten times more variable

per site Three times more polymorphisms than HV alone

http://www.sas.upenn.edu/~tgschurr/labwork/labwork_text.html

Current Method

• Sequence based on chromatogram

• Consensus of one forward and one reverse

NGS

• Sequence based on thousands of individual reads

• Improved sensitivity: – Mixture detection

– Low level heteroplasmy

mtDNA Information

Current Method

• Minor peaks may not be reproducible

• SRM 2392 9947a, 1393 G/A heteroplasmy

NGS

• More consistent detection of minor genotypes

• Validation important – Variant calling thresholds

– Characterizing noise

mtDNA Information

Characterization of SRM 2392 and 2392-I Mitochondrial genome sequencing standard

Detection of low level heteroplasmy

0.0%

5.0%

10.0%

15.0%

20.0%

HL60 - 2445 HL60 - 5149 9947A - 1393 9947A - 7861

Min

or

Alle

le F

req

ue

ncy

C T T A

2878 1981 1571

571

18821

10884

22719

17499

41064 40356

48071

42101

11737

16411 17697

11796

0

10000

20000

30000

40000

50000

0.0%

5.0%

10.0%

15.0%

20.0%

HL60 - 2445 HL60 - 5149 9947A - 1393 9947A - 7861

Co

verage

M

ino

r A

llele

Fre

qu

en

cy

Characterization of SRM 2392 and 2392-I Mitochondrial genome sequencing standard

Detection of low level heteroplasmy

C T T A

SNaPshot

http://portal.ccg.uni-koeln.de/

Allelic Discrimination (qPCR)

Forensic DNA Markers






Sanger Sequencing

Most methods are low throughput and/or require a lot of DNA

NGS method can analyze many SNPs for many samples in one run

• IISNP-Individual

• AISNP-Ancestry

• LISNP-Lineage

• PISNP-Phenotype

SNP Information

• Individual Identification

– Balancing has occurred in all populations

– Low F statistics within (FIS) and among (FST) populations

– High heterozygosity

SNP Information

• Individual Identification Pakstis 2010, Kidd 2012

– Panel of 45 unlinked SNPs

– FST below ≈ 0.07

– Avg het > 0.4

– RMP 10-15 to 10-18

in 44 populations

SNP Information

• HID-Ion Ampliseq Identity Panel (version 2.3)

– 90 autosomal SNPs

– 30 Y-chromosome SNPs

– RMP 10-35

SNP Information

Kidd 45

SNPforID 52

HID Identity Panel

• Ancestry Information – High Fixation Index (FST)

– Population specific fixation has occurred

– Low heterozygosity

• Example – Malaria resistance SNPs in

Sub-Saharan Africa

SNP Information

• HID Ancestry Panel

– Beta version 3.0

– Publicly available soon

– 170 loci

– Derived from

Kosoy et. al (2008): 128 SNPs Kidd et. al (2014): 55 SNPs

SNP Information

• Ion Torrent Personal Genome Machine (PGM) – Launched in 2010

• Ion Torrent sequencing: – Emulsion PCR for single copy reactors

– Non-labeled nucleotide triphosphates

– Flowed over a bead on a semiconductor surface

• Hydrogen Ion detection – pH change is detected

– No optics

Life Tech - Ion Torrent - PGM

Ion Torrent PGM Workflow

http://www.youtube.com/watch?v=MxkYa9XCvBQ

The PGM Instrument at NIST

PGM Sequencer

OneTouch 2 (Emulsion PCR)

OneTouch ES (Enrichment)

7 ft

Ampliseq Workflow

PCR amplify

Chew back primers

Ligate adapters

Emulsion PCR

Sequencing

One template per bead/droplet

454 PGM “Ionogram”

Ion Ampliseq Library Kit

1 ng DNA input

Ampliseq Primer Pool

Template Kit Sequencing Kit

Front-End: Multiplex PCR

• HID-Ion Ampliseq Identity Panel (IISNP) – 120 markers in a single PCR reaction – Amplified regions 33 bp to 192 bp long

• HID-Ion Ampliseq Ancestry Panel (AISNP)

– 170 markers in a single PCR reaction – Amplified regions 34 bp to 136 bp long

• Small amplicons well suited to degraded or damaged DNA

60° 4:00

99° 2:00

99° 0:15

18 Cycles Time ≈ 1:30

4°

∞

1 ng DNA

Digest Primer Regions & Ligate Adaptors

• Enzymatic digestion removes ≈ 25 bp from ends of amplicons

• Universal sequencing adaptors are ligated to DNA – Adaptors termed P1 and A

• Barcoded sequencing adaptors can be used in this step

– Sequence multiple samples in one PGM run

P1 Adaptor

A Adaptor

Barcode Sequence

PCR Fragment

Adapted and Barcoded Sequencing Template

Enzyme Ligase

Prepare Ion Sphere Particles (ISPs)

• Libraries quantified by qPCR – Quantity of DNA going into emPCR is very important! – Goal: 10 % to 30 % template positive ISPs

• Too much DNA polyclonal ISPs (mixed read)

• Emulsion PCR

– Nanoliter droplets of PCR reagents in oil – Attaches a single DNA molecule to a single ISP

• Enrich for positive ISPs

– Liquid handler removes non-templated ISPs – Biotinylated primer/streptavidin beads

Ideal Non- Ideal




• Emulsion PCR

– Nanoliter droplets of PCR reagents in oil – Attaches sequencing template to the ISP



Ideal Non- Ideal

OneTouch 2




• Emulsion PCR

– Nanoliter droplets of PCR reagents in oil – Attaches sequencing template to the ISP



Ideal Non- Ideal

OneTouch 2

ISP

Magnetic bead w/ Streptavidin

Biotinylated PCR product

ISP NaOH

OneTouch ES

Sequencing & Data Analysis

• Library ISPs loaded onto chip

• PGM runs flows & detects pH

• Torrent Server & Torrent Suite Software – Processes pH signal into base calls

– Displays run summary

– Maps reads to reference genome Photo: www.lifetechnologies.com

Data Analysis HID SNP Genotyper Plugin

Allele coverage histogram

Normalized y-axis scale

X-axis is refSNP I.D.

Autosomal SNPs Y-SNPs

Data Analysis HID SNP Genotyper Plugin

Total Coverage

Reads for Each Base

Coverage for Either Strand

Strand Bias

Genotype

Quality Score

Major Allele Frequency

HID SNP Panel Sensitivity Study

• Dynamic range of DNA input to PCR – 1 ng is recommended – 10 ng (1 data point) – no problems were observed – 1 ng – 0.5 ng – 0.1 ng – 0.05 ng

• Libraries were generated and pooled (n = 12) • Sequenced on PGM 318 chip (11 M wells)

– 200 bp read chemistry

3 Replicates

0.5 ng Input DNA

HID SNP Panel Sensitivity Study A

ll s

cale

d t

o 8

00

0X

co

vera

ge

90 Autosomal SNP loci, sorted from highest to lowest coverage

0.1 ng Input DNA

0.05 ng Input DNA

Thresholds: analytical = 50 RFU, stochastic = 300 RFU, PHR = 0.5

Thresholds:

50X analytical

300X stochastic

50% balance

0.05 ng Input DNA

0.1 ng Input DNA

0.5 ng Input DNA

Identifiler® Plus amplification (29 cycle), 25 µl reaction , 3500xl electrophoresis, 1.2 kV for 8 seconds Thresholds: analytical = 50 RFU, stochastic = 200 RFU, PHR = 0.5

All

sca

led

to

40

00

RFU



D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 D5S818 FGA 1 in

0.05 ng

1.78E+01

1.17E+05

1.17E+06

0.1 ng

4.35E+03

6.19E+10

6.34E+11

0.5 ng

5.67E+14

rs1

00

55

33

rs1

00

92

49

1

rs1

01

52

50

rs1

02

41

16

rs1

03

18

25

rs1

04

88

71

0

rs1

04

95

40

7

rs1

05

80

83

rs1

07

73

76

0

rs1

07

76

83

9

rs1

10

90

37

rs1

29

97

45

3

rs1

32

18

44

0

rs1

33

58

73

rs1

35

53

66

rs1

35

76

17

rs1

36

02

88

rs1

38

23

87

rs1

41

32

12

rs1

45

43

61

rs1

46

37

29

rs1

49

04

13

rs1

49

32

32

rs1

49

85

53

rs1

52

35

37

rs1

52

84

60

rs1

59

60

6

rs1

73

64

42

rs1

82

13

80

rs1

87

25

75

rs1

88

65

10

rs1

97

92

55

rs2

01

62

76

rs2

04

04

11

rs2

04

63

61

rs2

07

68

48

rs2

11

19

80

rs2

14

95

5

rs2

21

95

6

rs2

26

93

55

rs2

29

29

72

rs2

34

27

47

rs2

51

93

4

rs2

83

07

95

rs2

83

17

00

rs3

21

19

8

rs3

38

88

2

rs3

54

43

9

rs3

78

09

62

rs4

28

84

09

rs4

30

04

6

rs4

36

42

05

rs4

45

25

1

rs4

53

00

59

rs4

84

70

34

rs5

60

68

1

rs5

76

26

1

rs6

44

47

24

rs6

81

12

38

rs6

95

54

48

rs7

04

11

58

rs7

17

30

2

rs7

19

36

6

rs7

22

09

8

rs7

22

29

0

rs7

27

81

1

rs7

33

16

4

rs7

35

15

5

rs7

37

68

1

rs7

40

59

8

rs7

40

91

0

rs7

52

03

86

rs7

70

47

70

rs8

26

47

2

rs8

73

19

6

rs8

76

72

4

rs8

91

70

0

rs9

01

39

8

rs9

07

10

0

rs9

14

16

5

rs9

64

68

1

rs9

87

64

0

rs9

90

59

77

rs9

93

93

4

rs9

95

11

71

1 in

0.05 ng 6.12E+15

3.62E+18

7.17E+21

0.1 ng 7.58E+27

8.87E+27

2.88E+30

0.5 ng 1.16E+35

2.31E+35

Identifiler Plus

PGM HID SNP Panel v2.3

Just like STR loci, some SNPs are

consistently less robust

1 in

0.05 ng 6.12E+15

3.62E+18

7.17E+21

0.1 ng 7.58E+27

8.87E+27

2.88E+30

0.5 ng 1.16E+35

2.31E+35

1 in

0.05 ng

D8S1179 D3S1358 D2S1338 D19S433 vWA TPOX D18S51 FGA TH01 D5S818 D7S820 D16S539 CSF1PO D21S11 1.78E+01

D8S1179 vWA D18S51 TH01 D5S818 D7S820 D16S539 CSF1PO D21S11 1.17E+05

D19S433 TPOX FGA TH01 D5S818 D7S820 D16S539 CSF1PO D21S11 1.17E+06

0.1 ng

D2S1338 D19S433 vWA TPOX D18S51 FGA D7S820 D16S539 CSF1PO D21S11 4.35E+03

D2S1338 D21S11 6.19E+10

CSF1PO D21S11 6.34E+11

0.5 ng

5.67E+14


Identifiler Plus

PGM HID SNP Panel v2.3

SNPs have more possible loci and better

performance at low levels


1

1000

1000000

1E+09

1E+12

1E+15

1E+18

1E+21

1E+24

1E+27

1E+30

1E+33

1E+36

0.05 ng 0.1 ng 0.5 ng

PGM HID SNP Panel Identifiler Plus

Ran

do

m M

atch

Pro

bab

ility

1

in

HID SNPs give better RMP

with 50 pg than ID+ gives with

0.5 ng

HID SNP Panel Sensitivity Study Summary

• Higher RMPs are expected for SNP panel compared to STRs due to many more loci

• Under thresholds indicated, higher % SNPs produce results than STRs also

• Better STR assays (GlobalFiler or NGS-STR) may lessen the “gap”

• Validation needed for SNP thresholds

HID SNP Panel Degraded DNA Study

Sheared genomic DNA

→Covaris S2 Focused Ultrasonicator

+ =

gDNA Sheared DNA


Sheared DNA was fractionated by size range

Blue Pippin system (3% Gel)

Automated size selection

1) 50 bp to 200 bp

2) 50 bp to 150 bp

3) 50 bp to 100 bp

4) 50 bp to 75 bp

5) 35 bp to 50 bp

Five individual agarose columns

Size fractionated fragments collected into recovery wells

1 2 3 4 5


Sheared DNA was fractionated by size range Agilent Bioanalyzer Trace

Size selected sheared DNA 50 bp to 200 bp

50 bp to 150 bp

50 bp to 100 bp

50 bp to 75 bp

35 bp to 50 bp

Input to HID Panel PCR 1 ng DNA

Built libraries and sequenced

Bioanalyzer Standard

Blue Pippin Marker (65 bp)


90 autosomal IISNPs

HID SNP Panel

Fragmented, size selected < 75 bp




Fragmented, non-size selected

Minifiler® amplification (30 cycle), 25 µl reaction, 3500xl electrophoresis, 1.2 kV for 8 seconds Thresholds: analytical = 100 RFU, PHR = 0.5; data scaled to 1000 RFU



Performed in

triplicate

One rep shown





Fragmented, non-size selected

PG

M 3

18

Ch

ip, a

ll s

cale

d t

o 2

00

0X

co

vera

ge


90 Autosomal SNPs, sorted from smallest to largest

Thresholds:

50X analytical

300X stochastic

50% balance

RMP 1 in

rs10

055

33

rs10

092

49

1

rs10

152

50

rs10

241

16

rs10

285

28

rs10

318

25

rs10

488

71

0

rs10

495

40

7

rs10

580

83

rs10

773

76

0

rs10

776

83

9

rs11

090

37

rs12

997

45

3

rs13

218

44

0

rs13

358

73

rs13

553

66

rs13

576

17

rs13

602

88

rs13

823

87

rs14

132

12

rs14

543

61

rs14

637

29

rs14

904

13

rs14

932

32

rs14

985

53

rs15

235

37

rs15

284

60

rs15

960

6

rs17

364

42

rs18

213

80

rs18

725

75

rs18

865

10

rs19

792

55

rs20

162

76

rs20

404

11

rs20

463

61

rs20

562

77

rs20

768

48

rs21

119

80

rs21

495

5

rs22

195

6

rs22

693

55

rs22

929

72

rs23

427

47

rs25

193

4

rs28

307

95

rs28

317

00

rs32

119

8

rs33

888

2

rs35

443

9

rs37

809

62

rs42

884

09

rs43

004

6

rs43

642

05

rs44

525

1

rs45

300

59

rs48

470

34

rs56

068

1

rs57

626

1

rs64

447

24

rs68

112

38

rs69

554

48

rs70

411

58

rs71

730

2

rs71

936

6

rs72

209

8

rs72

229

0

rs72

781

1

rs72

917

2

rs73

316

4

rs73

515

5

rs73

768

1

rs74

059

8

rs74

091

0

rs75

203

86

rs77

047

70

rs82

647

2

rs87

319

6

rs87

672

4

rs89

170

0

rs90

139

8

rs90

710

0

rs91

416

5

rs91

711

8

rs93

828

3

rs96

468

1

rs98

764

0

rs99

059

77

rs99

393

4

rs99

511

71

75-1 1.17E+01

75-2 3.14E+01

75-3 3.14E+01

100-1 6.83E+05

100-2 2.92E+07

100-3 1.38E+08

150-1 1.05E+21

150-2 8.75E+19

150-3 5.08E+20

200-1 1.96E+30

200-2 3.25E+26

200-3 6.61E+27

250-1 1.75E+36

250-2 8.62E+35

250-3 3.47E+36


PG

M II

SNP

s (9

0)

Min

iFile

r ST

Rs

(8)

RMP 1 in

PG

M II

SNP

s (9

0)

Min

iFile

r ST

Rs

(8)

RMP 1 in


SNPs have MANY more possible loci

and better performance in

degraded samples

75-1

75-2

75-3

100-1

100-2

100-3

150-1 7.08E+03

150-2 7.08E+03

150-3 1.26E+03

200-1 7.77E+07

200-2 6.94E+09

200-3 6.94E+09

250-1 6.94E+09

250-2 6.94E+09

250-3 6.94E+09

RMP 1 in

1.0E+00

1.0E+03

1.0E+06

1.0E+09

1.0E+12

1.0E+15

1.0E+18

1.0E+21

1.0E+24

1.0E+27

1.0E+30

1.0E+33

1.0E+36

1.0E+39


HID SNP Panel Degraded DNA Study Summary

• SNPs and STRs show expected performance in each fraction based on amplicon size

• Some SNPs can still amplify in degraded samples where STRs cannot

• Due to the high number of SNPs, very high RMPs are possible

• Better STR assays (GlobalFiler or NGS-STR) may lessen the “gap”

• Validation needed for SNP thresholds

SNPs and Mixtures

AA

AB

BB

1 A B

AA 100% 0%

AB 50% 50%

BB 0% 100%

% is coverage (like PH balance)

50

55

60

65

70

75

80

85

90

95

100

???

HID SNP Panel Mixture Detection

Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

HOMOZYGOTE AA or BB

HETEROZYGOTE AB

Single source

samples should be either

50% or 100%

One single source

sample, major allele frequency plotted

for 90 HID SNPs (in ascending order)

Akin to an imbalanced STR locus

50

55

60

65

70

75

80

85

90

95

100


Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

HOMOZYGOTE AA or BB

HETEROZYGOTE AB

Single source


50% or 100%

One single source sample in triplicate,

major allele frequency plotted


3 SNPs give outlying values, less useful for

mixtures

50

55

60

65

70

75

80

85

90

95

100


Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

HOMOZYGOTE AA or BB

HETEROZYGOTE AB

Single source


50% or 100%

Two single source samples in triplicate,



50

55

60

65

70

75

80

85

90

95

100


Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

HOMOZYGOTE AA or BB

HETEROZYGOTE AB

Single source


50% or 100%

Three single source samples in triplicate,



50

55

60

65

70

75

80

85

90

95

100


Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

HOMOZYGOTE AA or BB

HETEROZYGOTE AB

Single source


50% or 100%

Four single source samples in triplicate,



50

55

60

65

70

75

80

85

90

95

100


Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

HOMOZYGOTE AA or BB

HETEROZYGOTE AB

Single source


50% or 100%

Five single source samples in triplicate,



Assessing single source outliers

will improve mixture model

1 1 1A 1A 1B 1B A B

AA AA 2 2 0 0 100% 0%

AA AB 2 1 0 1 75% 25%

AB AA 1 2 1 0 75% 25%

AA BB 2 0 0 2 50% 50%

AB AB 1 1 1 1 50% 50%

BB AA 0 2 2 0 50% 50%

AB BB 1 0 1 2 25% 75%

BB AB 0 1 2 1 25% 75%

BB BB 0 0 2 2 0% 100%

SNPs in 1:1 Mixtures

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5 6 7 8 9

B

A

AA AA

AA AB

AB AA

AA BB

AB AB

BB AA

AB BB

BB AB

BB BB

1 1

50

55

60

65

70

75

80

85

90

95

100

Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs

A two-person

mixture in a 1:1 ratio should have

frequencies at: 50%, 75%, and 100%


One single source



Theoretical distribution— Size of “bin” is related to average heterozygosity

Genotype combinations in this bin are:

AA:AB, AB:AA, AB:BB, BB:AB

AA:BB, AB:AB, BB:AA

AA:AA, BB:BB

2 1 2A 1A 2B 1B A B

AA AA 4 2 0 0 100% 0%

AA AB 4 1 0 1 83% 17%

AA BB 4 0 0 2 67% 33%

AB AA 2 2 2 0 67% 33%

AB AB 2 1 2 1 50% 50%

AB BB 2 0 2 2 33% 67%

BB AA 0 2 4 0 33% 67%

BB AB 0 1 4 1 17% 83%

BB BB 0 0 4 2 0% 100%

AA AA

AA AB

AA BB

AB AA

AB AB

AB BB

BB AA

BB AB

BB BB

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5 6 7 8 9

B

A

2 1


50

55

60

65

70

75

80

85

90

95

100

Maj

or

Alle

le F

req

ue

ncy

90 Autosomal SNPs


A two-person mixture in a 1:1

ratio should have frequencies at: 50%, 67.5%, 82.5%, 100%

One single source



3 1 3A 1A 3B 1B A B

AA AA 6 2 0 0 100% 0%

AA AB 6 1 0 1 88% 13%

AA BB 6 0 0 2 75% 25%

AB AA 3 2 3 0 63% 38%

AB AB 3 1 3 1 50% 50%

AB BB 3 0 3 2 38% 63%

BB AA 0 2 6 0 25% 75%

BB AB 0 1 6 1 13% 88%

BB BB 0 0 6 2 0% 100%

AA AA

AA AB

AA BB

AB AA

AB AB

AB BB

BB AA

BB AB

BB BB

3 1

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5 6 7 8 9

B

A


50

55

60

65

70

75

80

85

90

95

100

Var

ian

t Fr

equ

en

cy

90 Autosomal SNPs


ratio should have frequencies at:

50%, 62.5%, 75%, 87.5% and 100%

One single source




50

55

60

65

70

75

80

85

90

95

100

>3:1 or >2 people =

>> “bins”

Var

ian

t Fr

equ

en

cy

90 Autosomal SNPs




50%, 62.5%, 75%, 87.5% and 100%

One single source sample and one 3:1

mixed sample, major allele

frequency plotted for 90 HID SNPs

(in ascending order)

Actual vs Theoretical

50

55

60

65

70

75

80

85

90

95

100

Var

ian

t Fr

equ

en

cy

90 Autosomal SNPs



50%, 62.5%, 75%, 87.5% and 100%

One single source sample and two 3:1

mixed samples, major allele




50

55

60

65

70

75

80

85

90

95

100

Var

ian

t Fr

equ

en

cy

90 Autosomal SNPs



50%, 62.5%, 75%, 87.5% and 100%

One single source sample and three 3:1

mixed samples, major allele




HID SNP Panel Mixtures Summary

• Mixtures can be detected in SNP data based on the coverage levels at heterozygous loci

• It may be possible to determine two-person 1:1 or 2:1 mixtures (maaaybe 3:1)

• More than two contributors or greater than 3:1 mixtures will be difficult to distinguish

• Need to determine which SNPs “behave”

• Stay tuned!

PGM AIM Panel (beta testing)

• Ampliseq library prep

• 170 SNPs

• Seldin 128

• Kidd 55

• Analysis plug-in integrates FROGkb

AIM Panel Ancestry Prediction – SRM 2391c

SRM 2391c Component

Gender Ethnicity

(self declared)

A Female Not listed

B Male Mexican-American

C Male Melanesian

D Female:Male Mixed sample

E Female Not listed

F Male Caucasian

• Likelihood Ratio calculations – Four categories extant in both Kidd and Seldin studies

• Europeans, African Americans, Maya, and Han Chinese

– Allows comparison of SNP sets’ performance – Representative of major U.S. populations

HID SNP Genotyper Plugin (v4.1 Beta) New Feature – Ancestry Map

• Heatmap of highest probability of origin

Ancestry Prediction SRM 2391c Component A

SRM 2391c Component

Gender Ethnicity Kidd 55

Prediction Seldin 128 Prediction

A Female Not listed European 1.02 x 1033

European 6.32 x 1066

Kidd 55 SNPs Seldin 128 SNPs

Ancestry Prediction SRM 2391c Component B

SRM 2391c Component



B Male Mexican-American


Han Chinese 1.48 x 1019


Ancestry Prediction SRM 2391c Component C

SRM 2391c Component



C Male Melanesian Han Chinese 1.54 x 1014

Han Chinese 6.67 x 1028


Ancestry Prediction SRM 2391c Component E

SRM 2391c Component



E Female Not listed European 5.41 x 1021



Ancestry Prediction SRM 2391c Component F

SRM 2391c Component



F Male Caucasian European 2.35 x 1031



HID SNP Panel Ancestry Summary

• 170 SNP panel containing two SNP sets that are suitable for use in U.S.

• Plug-in integrates FROG-kb (http://frog.med.yale.edu/FrogKB/)

• Heat maps give quick overview

• Interpretation tools being developed

– Combining loci

– Choosing/combining populations

http://frog.med.yale.edu/FrogKB/http://frog.med.yale.edu/FrogKB/

Conclusions

• NGS can give more information on currently used forensic markers

– More STRs and STR sequence info

– Whole genome mtDNA

• NGS facilitates genotyping of forensic SNPs

• SNPs may help with low level & degraded samples

• SNPs may provide ancestry (and phenotype?) information

• Forensic NGS kits/methods are being developed

• Many questions to answer prior to implementation

Acknowledgements

Dr. Peter Vallone Group Leader

Kevin Kiesler Research Biologist

THANK YOU

Funding from the FBI Biometrics Center of Excellence

Forensic DNA Typing as a Biometric Tool

Thermo Fisher (Life Tech):

Nnamdi Ihuegbu

Robert Lagace

Applied

Genetics Thank you for your attention!

Contact Info:

[email protected]

301-975-6401
mailto:[email protected]

Documents

Next Generation Sequencing Activities at NIST · 2020. 5. 5. · •HID-Ion Ampliseq Identity Panel (IISNP) –120 markers in a single PCR reaction –Amplified regions 33 bp to 192