Forensic Genetics:

Research Projects and 

Standards Production

Peter M. Vallone, Ph.D.

Leader, Applied Genetics Group

Forensics at NIST 2020

November 5, 2020
Disclaimer

• Points of view in this document are those of the author and do not necessarily 

represent the official position or policies of the U.S. Department of Commerce

• 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 endorsement by NIST, nor does it imply 

that any of the materials, instruments, or equipment identified are necessarily the 

best available for the purpose. 

• All work presented has been reviewed and approved by the NIST Research 

Protections Office.
DNA 

Extraction

DNA 

Quantitation

PCR 

Amplification

Genotyping & 

Sequencing Interpretation

Forensic Genetics – Forensic DNA Typing Workflow

Standards and 

data

Community 

Engagement

Applied 

research and 

assessing 

emerging 

technologies

Advancing technology and traceability through 

quality genetic measurements to aid work in 

Forensic and Clinical Genetics.

Variations upon the polymerase chain reaction (PCR) 

technique such as rapid PCR, multiplex PCR, realtime PCR, and digital PCR are used to genotype, 

sequence, and provide quantitative information

pertaining to an organism's genome.
Forensic Genetics Team

Margaret Kline will be retiring on November 20, 2020

Congratulations Margaret on a 35-year career at NIST

We’ll miss you!
Topics for today
SRM 2391 series

• SRM 2391c and 2391d – PCR-based DNA standard

• Used by the community to calibrate STR typing 

methods

• Purchased by practitioners and companies 

performing validations

• Further characterization of the samples and updates 

to the certificate

STR profile – certified allele calls
SRM 2391d

Certified and 

Information 

Values

Autosomal STR 

48 Markers + 

Amelogenin

Y-STR 

31 Markers

X-STR 

12 Markers

mtDNA

Whole Genome

Identity SNPs

94 ForenSeq

124 Precision ID 

Phenotypic SNPs

24 ForenSeq

24 Precision ID 

Ancestry SNPs

54 ForenSeq

168 Precision ID 

Markers included in the Certificate of Analysis

Indels

30 Markers

INNULS

20 Markers
• Next Generation Sequencing (NGS) was 

performed with two different 

instruments:

• MiSeq FGx (Verogen)

• Ion S5 XL (Thermo Fisher)

Platforms used for characterization

• Capillary Electrophoresis (CE) was 

performed with one instrument:

• 3500xL Genetic Analyzer (Thermo Fisher)

3500xl MiSeq FGx Ion S5 XL
Autosomal STR 

Marker List

MiniFiler

Identifiler

Identifiler Plus

Identifiler Direct

NGM

NGM SElectNGM Detect

Verifiler Plus

Verifiler Express

GlobalFiler

GlobalFiler Express

PP S5

PP CS7

PP 16

PP 16 HS

PP 18D

PP 21

PP ESX 17

PP ESX 17 Fast

PP ESI 17 Pro

PP ESI 17 Fast

PP Fusion

PP Fusion 6C

PP VersaPlex 27PY

ESSplex SE Plus

HDplex

24plex GO!

24plex QS

ForenSeq

Precision ID GF

PowerSeq 46GY

CODIS 20

European Standard Set

Certified Value

Information Value

D1S1656X

D1S1677X

D2S1338X

D2S441X

D2S1360X

D2S1776X

D3S1358X

D3S1744X

D3S4529X

D4S2366X

D4S2408X

D5S818X

D5S2500X

D5S2800X

D6S474X

D6S1043X

D7S820X

D7S1517X

D8S1132X

D8S1179X

D9S1122X

D10S1248X

D10S2325X

D12S391X

D12ATA63X

D13S317X

D14S1434X

D16S539X

D17S1301X

D18S51X

D19S433X

D20S482X

D21S11X

D21S2055X

D22S1045X

CSF1POX

F13A01X

F13BX

FESFPSX

FGAX

LPLX

Penta CX

Penta DX

Penta EX

SE33X

TH01X

TPOXX

vWAX

Autosomal STR Markers

Thermo Fisher CE STR kits

Promega CE STR kits

Qiagen Investigator CE STR kits

Verogen NGS kit

Thermo Fisher NGS kits

Promega NGS kits

CODIS 20/ESS 12

Autosomal STR Markers

35 Certified Autosomal STR Markers

13 Information Autosomal STR Markers

Materials characterized using 

various commercial kits
Autosomal STR Markers

Thermo Fisher CE STR kits

Promega CE STR kits

Qiagen Investigator CE STR kits

Verogen NGS kit

Thermo Fisher NGS kits

Promega NGS kits

CODIS 20/ESS 12

Autosomal STR Markers

35 Certified Autosomal STR Markers

13 Information Autosomal STR Markers

Autosomal STR 

Marker List

MiniFiler

Identifiler

Identifiler Plus

Identifiler Direct

NGM

NGM SElectNGM Detect

Verifiler Plus

Verifiler Express

GlobalFiler

GlobalFiler Express

PP S5

PP CS7

PP 16

PP 16 HS

PP 18D

PP 21

PP ESX 17

PP ESX 17 Fast

PP ESI 17 Pro

PP ESI 17 Fast

PP Fusion

PP Fusion 6C

PP VersaPlex 27PY

ESSplex SE Plus

HDplex

24plex GO!

24plex QS

ForenSeq

Precision ID GF

PowerSeq 46GY

CODIS 20

European Standard Set

Certified Value

Information Value

D1S1656 X

D1S1677 X

D2S1338 X

D2S441 X

D2S1360 X

Typed by CE methods NGS methods
Y

-STR Markers

28 Certified Y-STR Markers

3 Information Y-STR Markers

Y-STR 

Marker List

GlobalFiler

GlobalFiler Express

Yfiler

Yfiler PlusPP Fusion

PP Fusion 6C

PP VersaPlex 27PY

PowerPlex Y23

24plex GO!

24plex QS

ForenSeq

Precision ID GF

PowerSeq 46GY

Certified Value

Information Value

DYS19

X

DYS385a/bX

DYS389I/IIX

DYS390X

DYS391

X

DYS392X

DYS393X

DYS437X

DYS438

X

DYS439X

DYS448X

DYS449X

DYS456X

DYS458X

DYS460X

DYS461

X

DYS481X

DYS505

X

DYS518X

DYS522X

DYS533X

DYS549X

DYS570X

DYS576X

DYS612X

DYS627X

DYS635X

DYS643X

Y-GATA-H4X

DYS387S1X

Y-STR Markers

Thermo Fisher CE STR kits

Promega CE STR kits

Qiagen Investigator CE STR kits

Verogen NGS kit

Thermo Fisher NGS kits

Promega NGS kits
Information for additional marker systems

Support the adoption of new markers and technology platforms

• Mitochondrial genome sequence

• Identity SNPs – for degraded samples

• Ancestry SNPs – biogeographical ancestry prediction

• Phenotype SNPs – eye and hair color prediction
SNP allele calls for all components
How can SRM 2391d be used in YOUR lab?

• To meet the FBI Quality Assurance Standards: QAS 8.4

• Validation Studies: instrument, commercial kit, and software 

• Developmental and Internal Validations

• Known, well-characterized samples for forensic marker systems

• Make NIST traceable materials: 

http://ts.nist.gov/traceability/
Establishing Traceability to NIST SRM 2391d

• Traceability requires the establishment of an unbroken chain of 

comparisons to stated references: http://ts.nist.gov/traceability/

• In the case of DNA testing with STR markers, the reference material is 

SRM 2391d

• Materials deemed traceable to NIST-created materials must have a 

record associated with them

‘Lot’ of DNA material

Run against SRM 2391d

Everyday use calibrant

Contact becky.steffen@nist.gov for traceability questions
Notes for SRM 2391c and Mitochondrial SRMs

• For those still using SRM 2391c (no 

longer being sold) the certificate 

expiration date has been extended 

through February 3, 2022

• This will be the final extension, after 

that SRM 2391d must be used

• SRMs 2392 and 2392-I 

(mitochondrial DNA sequencing) 

will not be replaced – use SRM 

2391d 
Topics for today
SRM 2372a (Human DNA Quantitation Std)

• SRM 2372a – Human DNA Quantitation Standard

• Used to calibrate qPCR methods and commercial 

DNA standards

• Digital PCR used for value assignment

• Mitochondrial to nuclear DNA ratio information 

included

https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP.260-189.pdf

Male

Female

1:3 M/F
Digital PCR

Partitioning of DNA targets into individual chambers or droplets

dPCR is counting accessible amplifiable targets

Positives 

(1 copy of the target)

Negatives 

(0 copies of the target)

A standard curve is 

not needed
These are the assigned values 

(with error) for the 

Components in SRM 2372a

Value and uncertainty based 

on ten unique digital PCR assays

Assigned concentration values

A B C

[DNA] ng/μL

25

30

35

40

45

50

55

60

65

Value for SRM

Value for SRM

Value for SRM

SRM 2372a Component 

10%
SRM 2372a includes the ratio of mitochondrial 

to nuclear haploid genomes

mtDNA/nDNA ratio for three 

mitochondrial quantification assays 

optimized for dPCR

Developing Y chromosome specific 

digital PCR assays
Topics for today
STRBase and STRBase 2.0

https://strbase-b.nist.gov/

https://strbase.nist.gov/
Updated navigation of STR fact 

sheets and variant alleles

Updated topic pages for:

• SNP markers

• Mitochondrial DNA

• Insertion-Deletion markers

• Forensic SRMs

• NIST Interlaboratory studies

• Population data

• Educational resources

• Please visit and give us any 

feedback: strbase@nist.gov

https://strbase-b.nist.gov/

STRBase 2.0
Forensic DNA Open Dataset

• Periodic requests for raw CE (.HID) or sequencing files (.FASTQ)

• From academics, researchers, software vendors, etc

• Goal: make data freely available for learning/training purposes

• Source: Eleven single source samples previously screened as 

SRM candidates (not NIST population samples or SRM samples)

https://data.nist.gov/od/id/mds2-2157

Email Katherine.Gettings@nist.gov

...for questions/ideas
Topics for today
Rapid DNA Maturity Assessment

What is “Rapid DNA”? 

A fully automated instrument capable of generating 

a DNA profile from a swab (‘swab in – profile out’)

The maturity assessment was an interlaboratory 

study assessing DNA typing success and accuracy 

results on three Rapid DNA platforms

Twenty swabs were provided to each participant 

(crime labs, police agencies, vendors)

The results support the implementation of Rapid 

DNA instrumentation at the booking stations for 

single source samples

Stakeholders: FBI laboratory, DNA databasing labs, 

booking stations, Rapid DNA community, ANDE, 

Thermo Fisher
Assessment Scheme

NIST provides 20

reference buccal 

swabs to each 

participant

Participant Runs 

RH-ID GFE

Participant Runs 

RH200 GFE

Participant Runs 

ANDE FlexPlex

NIST reports 

CODIS 20 success 

rate for all data 

combined 

(% success)

• Participants may choose one chemistry per 20 NIST 

provided swabs

• Additional packages may be requested

Data transferred back to NIST via 

electronic format
Profiles from high quality single source samples generated by Rapid DNA instruments
CODIS 20: 85%

Full Profile: 80%

CODIS 20CODIS 20

Full ProfileFull Profile

40

60

80

100

Success (%)

Genotyping Success: Rapid DNA Analysis

Each data point is an 

independent instrument
CODIS 20: 90%

Full Profile: 90%

CODIS 20CODIS 20

Full ProfileFull Profile

40

60

80

100

Success (%)

Genotyping Success: Modified Rapid DNA Analysis

Each data point is an 

independent instrument
Topics for today
Examination of Front-End Methods in 

DNA Typing

• Problem: Assess the amount of sample loss during the extraction 

• Low extraction efficiency cold result in overall lower sample quantity

• May fail to yield full STR profiles or minor components in mixtures

Methods for determining extraction efficiency and sample loss vary

0.5 ng

Starting DNA amount

100 pg

Ending DNA Amount

Extraction

Sample Loss
Absolute Extraction Efficiency

Offers the ability to evaluate individual extraction processes and 

their efficiency independent of another method

DNA Recovered

Original Amount DNA

Absolute Extraction 

Efficiency

Measured by digital PCR
DNA Sources

Component A of SRM 2372a: 

Human DNA Quantitation 

Standard 

Known concentration of 49.8 ng/μL

Determined by digital PCR

Freshly collected whole 

blood

Known WBC of 4.6 x103 per μL

WBC reported by blood bank

Washed cell suspension in 

dPBS

Known cell count of 1x106 per mL

Determined by flow cytometry
DNA Extraction Methods

QIAamp Spin Columns

• Manual method commonly used 

in forensic DNA laboratories

• Silica columns for collection of 

DNA

• Elution in proprietary buffer 

• Similar to TE-4

Qiagen EZ1 Advanced XL

• Robotic purification instrument

• Cell lysis takes place on the 

benchtop in a thermomixer

• Purification with paramagnetic 

bead collection

• Elution in TE-4

Phenol Chloroform (Organic)

• Often referred to as the 

“gold standard” 

• Proteinase K digestion of 

the cells

• Equal volumes of Phenol 

Chloroform added 

• Phase lock gel tubes used 

for promoting separation

• DNA was precipitated with 

Ethanol and resolubilized 

with TE-4 buffer

Extraction methods available in our lab
Four DNA input amounts were tested in replicates of 

five for each extraction method

60 Samples per DNA Source

60 Samples per Extraction Method

Amount (ng) # of Cells Uncertainty 

(± # Cells)

# of 

Replicates

50 8,333 833

20 3,333 333

10 1,667 167

5 781 78

38 6,250 313

19 3,125 156

9 1,563 78

5 781 39

276 46,000 2,300

138 23,000 1,150

28 4,667 233

14 2,333 117

Extracted 

DNA 

Blood

Cells

5 per amount 

(20 per DNA Source)

5 per amount 

(20 per DNA Source)

5 per amount 

(20 per DNA Source)
DNA Source

Extraction Method

Digital PCR was used to 

determine the concentration 

post-extraction 

The efficiency of the 

extraction methods are 

shown between methods 

and DNA sources

Repeating – re-optimizing

Romsos, E.L & Vallone, P.M. (2019) Estimation of extraction efficiency by droplet digital PCR. Forensic Sci Int Genet. Sup. Ser. DOI: 10.1016/j.fsigss.2019.10.072
Topics for today
DNA 

Extraction

DNA 

Quantitation

PCR 

Amplification

Genotyping & 

Sequencing Interpretation

DNA Mixture Interpretation

measurement

Statistics

Assign a strength of evidence

Hp Likelihood ratio (LR) : the DNA from the POI IS in the mixture

Hd: the DNA from the POI IS NOT in the mixture

I: background information

E: evidence

LR system
• Examine methods to assess 

the performance of LR 

systems using publicly 

available ground truth data 

(mixture profiles)

• Examine the similarities and 

differences between the LR 

systems

DNA Mixture Interpretation

Alfonse, L.E., et al. A large-scale dataset of single and mixed-source short tandem repeat profiles to 

inform human identification strategies: PROVEDIt. Forensic Sci. Int. Genetics 32, 62-70.

https://www.strmix.com/ http://www.euroformix.com/
Hp true STRmix

Hd true STRmix

Hp true EFM 

Hd true EFM 

Log10(LR) Distribution by PG model, NOC, & Propositions

Log10(LR)

2 Person 3 Person

Discrimination power of LR Systems using Hp true 

& Hd true LR distribution Known contributor Known non-contributor
Log10(LR) Distribution by Software & Mixture Ratios

Log10(LR) Distribution for 2P by Software & 

Proposition

Log10(LR)

Hp true STRmix Hd true STRmix Hp true EFM Hd true EFM Log10(LR)

Log10(LR) Distribution by Software & Mixture Ratios (2P)

1;1 1;2 1;4 1;9

STRmix EFM STRmix EFM STRmix EFM STRmix EFM

2P
Log10(LR) Distribution by Software & Treatment

Log10(LR) Distribution for 2P by Software & 

Proposition

Log10(LR)

Log10(LR) Distribution by Software & Treatment (2P)

Log10(LR)

Pristine DNase I UV Sonication Inhibition

STRmix EFM STRmix EFM STRmix EFM STRmix EFM STRmix EFM

Hp true STRmix Hd true STRmix Hp true EFM Hd true EFM 

2P
Global profile Log10(LR) from 2P and 3P for Hp true 

STRmix Log10(LR)

EFM Log10(LR)

2P

STRmix Log10(LR)

EFM Log10(LR)

Factor of 102

Factor of 104

Factor of 106

Factor of 102

Factor of 104

Factor of 106

2P 3P
Global profile Log10(LR) from 2P for Hp true 

STRmix Log10(LR)

EFM Log10(LR)

2P Factor of 102

Factor of 104

Factor of 106

2P

• In the process of finalizing 4-person mixtures

• Investigating the sources of variation

• 155 two-person mixtures

• 147 three-person mixtures

• 132 four-person mixtures

0

10

20

30

40

50

60

70

80

Relative frequency *100 ( n= 310)

Differences in log scale

Degree of agreement between LR systems 
Topics for today
Working group and stakeholder engagement

• NIST: Human Factors and Mixture Review

• NIJ FLNTWG – Sequencing white paper

• NIJ Technology Working Group

• FBI: SWGDAM

• Laboratory Operations, Sequencing, Body Fluid Identification

• FBI: Rapid DNA task groups

• ISFG: STR Nomenclature (Recommendations)

• NIST/ANSI Type 18 DNA Standard Working Group

• OSAC: Sequencing subgroup

• Pre-release testing for Promega, Thermo Fisher, QIAGEN

Working group and stakeholder engagement
Looking forward into FY21

• Skin protein work with IARPA (genetically variant peptides – GVP)

• Assessing the genetic genealogy landscape – needs for validation, further 

foundational research, and written standards 

• Probabilistic Modeling for Forensic Interpretation of DNA Mixtures Using 

Next Generation Sequencing Data

• The application of AI/machine learning for DNA mixtures (NoC, 

deconvolution)

• Y SNP interlaboratory study (Thermo Fisher, 800+ Y SNPs)

• Hosting a continuing education day at NIST

• Develop digital PCR assays for the Y chromosome

Into FY2021
Thank you for your attention

peter.vallone@nist.gov
Forensic Genetics:

Next Generation 

Sequencing

Katherine Butler Gettings, Ph.D.

Research Biologist, Applied Genetics Group

Forensics at NIST 2020

November 5, 2020
disclaimer

Points of view in this document are those of the author and do not 

necessarily represent the official position or policies of the U.S. 

Department of Commerce. 

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 

endorsement by NIST, nor does it imply that any of the materials, 

instruments, or equipment identified are necessarily the best available 

for the purpose. 

All work presented has been reviewed and approved by the NIST 

Research Protections Office.
Forensic Genetics Team

Margaret Kline will be retiring on November 20, 2020

Congratulations Margaret on a 35 year career at NIST

We’ll miss you!
Topics for today
Topics for today
Topics for today
Markers and peaks are separated by size (time)

Colors separated by fluorescent dye labels

Short Tandem Repeats
Single Nucleotide Polymorphism

Allele 1: TAGGATCGTGCCGATGACTG

Allele 2: TAGGATCGTACCGATGACTG

A/A A/G G/G

Homozygous Heterozygous Homozygous
Mitochondrial Genome

Topics for today
NGS Platforms @NIST

MiSeq FGx / RUO

• Verogen ForenSeq DNA Signature Prep

• Promega PowerSeq 46GY 

• Promega PowerSeq CRM Nested System 

• QIAseq Targeted Human Mitochondria Panel

• QIAgen GeneRead DNAseq Targeted V2 Panel (Identity SNP)

Ion Chef and Ion S5

• Precision ID GlobalFiler NGS STR Panel v2

• Precision ID Identity and Ancestry SNP Panels 

• Ion AmpliSeq DNA Phenotyping Panel

MinION

• mtDNA whole genome and microbial genome
Topics for today
Population Sample Sequencing

When a match is made in a forensic case, 

allele frequencies are used to calculate the rarity of the DNA profile 

8,9

2pq

2*0.144*0.375

0.108

1 in 9.3

Length

[ATCT]8, [ATCT]9

2pq

2*0.144*0.113

0.033

1 in 30.7

Sequence
Population Sample Sequencing

STR, Y-STR, X-STR and SNP

Illumina MiSeq FGx instrument, ForenSeq

• 27 autosomal STRs + 24 Y-STR + 7 X-STR + Amel

• 94 HID-SNPs + 56 ancestry SNPs + 22 phenotype SNPs

• 1036 Samples

• Sequenced in batches of 24 or 32

• 41 total sequencing runs in 2016
2018 

27 auSTR

2018

SE33

24 Y-STR

and 

94 HID-SNP

in progress

2020 7 X-STR

Population Sample Sequencing
2020 7 X-STR

Population Sample Sequencing
Population Data in NIST PDR
Population Data in NIST PDR
Mitochondrial Genome 

Population Sample Sequencing

659 samples attempted

• African American

• U.S. Caucasian

• U.S. Hispanic

704 samples attempted

• African American

• U.S. Caucasian

• U.S. Hispanic

• Native American

• Asian

Cassandra Taylor 

Kimberly Sturk-Andreaggi

Charla Marshall
Project Aims

Generate high-quality data

• CLC Bio Genomics Workbench Plugin 

• “AQME” developed by AFDIL

• Double review of variant calls

• Confirmation of heteroplasmy < 10 %

Phylogenetic QC by EMPOP

• International mtDNA database

• Identify unlikely variants

Large dataset of mtGenomes

• 1,327 total passed QC

• Searchable in EMPOP

• Enable match statistics (mtGenome)
Long-PCR Workflow

PCR

• Two long amplicons ~ 8.5 kb each

• QC +/- on AATI Fragment Analyzer

Library 

Preparation

• Fragmentation (enzymatic) of PCR products

• Ligate adaptors/barcodes

Sequencing

• QC & Quantitate libraries on AATI F/A

• Run on MiSeq FGx

Data Analysis

• Align to reference genome (rCRS)

• Dual Review QC of data

Origin

16,569  1

2,499

2,669

10,837 10,672
Dataset

Total 

Individuals

Total 

PHPs

Individuals 

with PHPs

Individuals 

with 1 PHP

Individuals 

with 2 PHPs

Individuals 

with 3 PHPs

COAF 112 37 31 (28 %) 26 4 1

COCN 112 41 30 (27 %) 20 9 1

COHS 109 36 27 (25 %) 20 5 2

NTAF 256 77 60 (23 %) 43 17 0

NTCN 260 92 77 (30 %) 65 10 2

NTHS 138 53 43 (31 %) 34 8 1

DSAS 169 62 54 (32 %) 49 2 3

DSNA 171 48 43 (25 %) 38 5 0

All 1327 446 365 (28 %) 295 60 10

Heteroplasmy Observed
Population Pairwise Fst Comparisons

• Similar population ‘samplings’

• African American and U.S. 

Caucasian

• Homogeneous across 

geographic samples

• Differences in U.S. Hispanics

• Could be due to site of 

collection

• Western U.S. vs Eastern
Topics for today
Combining Marker Types

We calculate Match Statistics by multiplying allele frequencies across markers

• Requires markers to be independent 

Sequencing allows typing more markers and different marker types

Laboratories need guidance on calculating the appropriate match statistics

Evaluating Linkage Disequilibrium in auSTR and IISNP loci in NIST 1036

• Collaboration with Andreas Tillmar, National Board of Forensic Medicine
TPOX

D3S1358

FGA

D5S818

CSF1PO

D7S820

D8S1179

TH01

vWA

D13S317

D16S539

D18S51

D21S11

D1S1656

D2S441

D2S1338

D10S1248

D12S391 

D19S433 

D22S1045

D6S1043

Penta D

Penta E

D4S2408

D9S1122

D17S1301

D20S482

CODIS 13 CODIS 7 Commonly Used

NIST Minis

27 Autosomal STRs 

in ForenSeq
44 from Kidd

50 from SNPforID

45 SNPs are spread across the 

22 human autosomes and show very loose 

or no genetic linkage with each other

LD tests for pairs of SNPs on same 

chromosome demonstrated no 

significant deviation from expectations

94 IISNPs in ForenSeq
LD in casework

When LD is detected, ideally:

• Rule out technical issues by testing on different platforms/assays

• Confirm with multiple sample sets from same population, and multiple test methods

Designing panel/assay: Evaluate LD, eliminate loci as needed based on informativeness

Implementing established panel/assay:

Best – Determine haplotype frequency for pair or block 

• for polymorphic loci the sample size would be unfeasible 

Alternative – Exclude one of the two markers during validation

• Keep the more informative, similar to assay design 

Problematic – Exclude one of the two markers case-by-case

• RMP vs Kinship
STRSeq Catalog of Sequences
NIST - 105

UNT - 80 USC - 96

KCL - 96

D12S391 Alleles by Lab

STRSeq Catalog of Sequences
https://www.ncbi.nlm.nih.gov

/bioproject/380127

STRSeq

Catalog of Sequences
STRSeq Catalog of Sequences
STRSeq Catalog of Sequences
STRSeq Catalog of Sequences
STRSeq in Population Data
Our mission is to harmonize related efforts across member laboratories:

STRAND working group align|name|define

and to 

characterize 

additional STR loci 

present in the genome 

which may be useful for 

forensic purposes in the future.

Population 

sample 

sequencing

Forensic STR 

Sequence 

Guide

STRait Razor

Bioinformatic 

freeware

STRSeq

Catalog of 

sequences

STRidER

STR sequence 

quality control
STR Nomenclature Meeting

April 2019

Spring 2020: 

ISFG EB approved 

STRAND WG proposal for 

DNA Commission on STR 

Sequence Nomenclature 

Recommendations

STR Nomenclature Meeting Report

Defined Coordinates

Supplementary File - 24 auSTRs

PowerSeq 46GY GeneMarker NGS Range

ForenSeq DNA Signature Prep Kit UAS Flanking Region Report Range

Precision ID GlobalFiler NGS v2 Converge .bed file range
Topics for today
Comparison of conventional CE versus NGS-STR genotyping workflows

Riman et al. 2020
RFU

D21S11 (29, 31)

Coverage in Log Scale

D21S11 (29, 31) Total Type of Sequences = 646 

Locus Coverage = 6949

A = True known allele sequences

A = True known allele sequences

S1 = Primary back stutter (LUS of basic repeat motif)

S1 S1

S2 = Back stutter sequences not attributed to S1

S2 S2

N = Noise sequences

N N N N

28

50 types

29

278 types

30

39 types

31

257 types 

Data Analysis by NGS

Data Analysis by CE

Alleles

Number of unique sequences
Average library concentration yield versus starting amount of DNA template

Riman et al. 2020
Average Locus Coverage relative to DNA template amounts

Normalized vs. Non-normalized libraries

Riman et al. 2020
Impact of DNA template amount on the distribution 

of known allele, stutter, and noise sequences

Riman et al. 2020
Topics for today
Y-SNP Sequence Study with 

U.S. Population Samples

Worldwide collaboration with Erasmus Medical Center Rotterdam
Call for Participants at ISFG 2019
Project Title: 

Bringing Forensic Y-Chromosome Haplogrouping to the Next Resolution Level 

by using Targeted Massively Parallel Sequencing

Study Organizers: 

Manfred Kayser and Arwin Ralf

Erasmus MC

Timeframe: Dec 2020 

(extended due to COVID-19)

Provided by Organizers

• Protocol

• Y-SNP Panel

Reference Paper
Description of Study – Sequencing Y-SNP markers

To obtain worldwide population frequency data:

• 884 Y-SNP Markers sequenced on Ion S5

• Infer 640 Y haplogroups

• ≥192 male samples per population

NIST 1032 male samples:

• 359 U.S. Caucasians

• 341 African Americans

• 236 U.S. Hispanics

• 96 U.S. Asians (extra data as needed)
Previous Study with Erasmus: Rapidly Mutating YSTR Markers with U.S. Population Male Samples
Topics for today
Future Directions

Implementation... what are the barriers?
Vendors create/adapt assays Developmental validation

and analysis methods

Casework Laboratories Internal validation

evaluate cost/benefit

Sequence Nomenclature Proficiency Tests

Probabilistic Databases 

Genotyping of the 

for NGS future

Future Directions

Implementation... what are the barriers?
Vendors create/adapt assays Developmental validation

and analysis methods

Casework Laboratories Internal validation

evaluate cost/benefit

Sequence Nomenclature Proficiency Tests

Probabilistic Databases 

Genotyping of the 

for NGS future

Future Directions

Implementation... what are the barriers?
Vendors create/adapt assays Developmental validation

and analysis methods

Casework Laboratories Internal validation

evaluate cost/benefit

Sequence Nomenclature Proficiency Tests

Probabilistic Databases 

Genotyping of the 

for NGS future

Sequence Nomenclature

Probabilistic 

Genotyping for NGS 

Proficiency Tests

Databases 

of the 

Future

Match Statistics

Future Directions

Implementation... what is needed?
Acknowledgements

Collaborators

David Ballard, Kings College London

Martin Bodner & Walther Parson, Medical University of Innsbruck 

Jonathan King, University of North Texas Health Science Center

Chris Phillips, University of Santiago de Compostela

George Riley, National Center for Biotechnology Information

Arwin Rolf & Manfred Kayser, Erasmus University Medical Center

Cassandra Taylor, Kimberly Sturk-Andreaggi, Charla Marshall, AFDIL

Andreas Tillmar, National Board of Forensic Medicine

Funding NIST Special Programs Office and the FBI Biometric Center of Excellence Unit: DNA as a Biometric.

Questions?

katherine.gettings@nist.gov

katherine.gettings@nist.gov