Population Sample 

Sequencing at NIST

FORENSICS@NIST – Forensic Genetics Session

9 November 2016

Katherine Butler Gettings, PhD

Research Biologist, Applied Genetics Group

Dr. Peter Vallone

Lisa Borsuk

Kevin Kiesler

Becky (Hill) Steffen
Official Disclaimer

The opinions and assertions contained herein are solely those 

of the author and are not to be construed as official or as views 

of the U.S. Department of Commerce.

Commercial equipment, instruments, software, or 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 the U.S. 

Department of Commerce, nor does it imply that any of the 

materials, instruments, software or equipment identified are 

necessarily the best available for the purpose.
Forensic DNA 

Current 

Technology:

Capillary 

Electrophoresis 

(CE)

Image courtesy Bruce McCord, Florida International University
Current 

Technology: 

CE Electro-

pherogram

Markers and peaks are separated by size (time)

Colors separated by fluorescent dye labels
CE Analysis

Internal Lane Standard

Allelic Ladder

Sample Genotype

D2S441 

Alleles 9-17

212 – 245 bp

D10S1248 

Alleles 8-19

250 – 294 bp

D13S317 

Alleles 5-17

297 – 345 bp

D2S441 

Sample is 10,14

D10S1248 

Sample is 13,15

D13S317 

Sample is 9,11

225 bp 250 bp 275 bp 300 bp 325 bp
100 bp 200 bp 300 bp 400 bp 500 bp

PowerPlex 1.1

c.1998

PowerPlex Fusion

c.2013

PowerPlex 35GY 8C

c.2017-18

CE Multiplex Evolution Examples

3 dye

8 locus

5 dye

24 locus

8 dye

35 locus

www.promega.com/products/genetic-identity/analysis/spectrum-ce-system-forensics-paternity
Advances in DNA Sequencing

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

Available at: www.genome.gov/sequencingcostsdata. Accessed 11/04/2016.

2001:

First human genome published, 

requiring 15 years of effort 

at a cost of 3 billion dollars

2014:

One instrument can sequence 

45 human genomes in one day 

for $1000 each
Sequencing Forensic STRs

• STR = Short Tandem Repeat

• Example [GGAA]13 

• Targeted sequencing reveals sequence 

variation within STR amplicons

• Greater degree of multiplexing

• Not confined by dye colors; smaller PCR amplicons

• Other loci (SNPs)
Promega PowerSeq Auto/YSTR/Mito System

Applied Biosystems Precision ID 

STR, SNP and Mixture Panels

Forensic NGS Kits

for STR and SNP Typing

Illumina ForenSeq

and FGx
• 27 autosomal STR loci

• 24 YSTR loci

• 7 XSTR loci

• Identity, Ancestry and Phenotype SNPs
ForenSeq

• 27 autosomal STR loci

When a match is made in a forensic case, allele frequencies are used to 

calculate how common or rare the DNA profile is in a given population
ForenSeq

• 27 autosomal STR loci

When a match is made in a forensic case, allele frequencies are used to 

calculate how common or rare the DNA profile is in a given population

Example data for illustration purposes only
At D4S2408, the length genotype is 9,10.

The Random Match Probability (RMP) is 2*p*q. 

p and q are the frequencies of 9 and 10.

The length-based RMP is 2pq = 2(0.375)(0.331) = 

0.248, meaning approximately 25% of individuals 

in this population would not be excluded as 

possible contributors.

Sequence Allele N Freq

[ATCT]7 1 0.6%

[ATCT]8 23 14.4%

[ATCT]9 18 11.3%

[ATCT] GTCT [ATCT]7 42 26.3%

[ATCT]10 53 33.1%

[ATCT]11 21 13.1%

[ATCT]12 2 1.3%

D4S2408

Allele N Freq

7 1 0.6%

8 23 14.4%

10 53 33.1%

11 21 13.1%

12 2 1.3%

9 60 37.5%

D4S2408

Example: Profile 

from crime scene is 

single source, and 

matches POI.

At D4S2408, the sequence genotype is [ATCT]9, [ATCT]10.

The associated statistic is 2*p*q. p and q are the 

frequencies of [ATCT]9 and [ATCT]10.

The sequence-based RMP is 2pq = 2(0.113)(0.331) = 

0.074, meaning approximately 7% of individuals in this 

population would not be excluded as possible 

contributors.

Example data 

for illustration 

purposes only
ForenSeq STR and IISNP Statistics

Length-based 

allele frequencies:
General workflow for 96 samples at NIST

PCR amp 

(DPMB) and 

tag STR loci

PCR enrich

adapters

barcodes

Purify 

libraries

Normalize 

libraries

Pool, 

denature 

and dilute 

libraries

Load FGx

Bridge PCR

Sequencing 

by synthesis

Afternoon of

Day 1

Morning of 

Day 2 Day 2

Afternoon of 

Day 2

-------------96 samples------------- ------------------ 3 x 32 samples-----------------

3

rd plate is loaded afternoon of day 6, 

96 samples complete on day 8

Day 2 Day 2

Finishes 

Morning of 

Day 4
Population Sample Sequencing Metrics

1036 Samples from four population groups

• 342 African American

• 361 Caucasian

• 97 East Asian

• 236 Hispanic

Sequenced in batches of 24 or 32

41 total sequencing runs 

Additional CE analysis to provide complete data for concordance check

Data Analysis, 

CE Backfilling

AAFS
Data analysis

• Length based genotypes from CE

• Sequences and length genotypes from UAS

• Tandem analysis with in-house pipeline based on:

Final allele sequences

SR

UAS CE

Illumina’s Universal Analysis Software
Slides have been removed 

from the original 

presentation, manuscript 

under preparation
• Many forensic STR loci contain underlying sequence variation 

• This will increase allelic diversity, thus increasing the ability to 

discriminate among individuals in a mixture

• NIST “1036” sequence-based allele frequencies support implementation

• CE concordance analysis ensures back-compatibility

Population Sample Sequencing

Conclusions
Acknowledgments

Funding

• NIST SPO - Forensic DNA

• FBI - DNA as a Biometric

• NIJ

Questions? katherine.gettings@nist.gov

Dr. Peter Vallone

Lisa Borsuk

Kevin Kiesler

Becky (Hill) Steffen