The Use of Statistical Analysis to Characterize Noise and 

Zygosity in Targeted Sequencing of Forensic STR Markers

Sarah Riman1, PhD; Hari Iyer2, PhD; Lisa Borsuk1, MS; Peter M. Vallone1, PhD

1Applied Genetics Group 

2Statistical Design, Analysis, and Modeling Group
Points of view in this presentation are mine and do not necessarily represent the

official position of the National Institute of Standards and Technology or the U.S.

Department of Commerce.

NIST Disclaimer Certain commercial products and instruments are identified in order to

specify experimental procedures as completely as possible. In no case does such an

identification imply a recommendation or endorsement by the National Institute of

Standards and Technology, nor does it imply that any of these products are necessarily

the best available for the purpose.

Disclaimer
Overview

▪ Forensic DNA typing using the gold standard “Capillary Electrophoresis” (CE) 

technology vs. “Next Generation Sequencing” (NGS) technology

▪ Why implement NGS if you can accomplish DNA typing by CE?

▪ Characterization of single-source PowerSeq 46GY DNA profiles 
Forensic DNA typing using the gold standard “Capillary Electrophoresis”

(CE) technology vs. “Next Generation Sequencing” (NGS) technology
Clonal Amplification/Cluster Generation

Sequence 

Data Analysis

General Workflow for Next Generation Sequencing (NGS)

DNA Extraction

DNA Quantitation

Targeted DNA Amplification Detection by CE Data Analysis 

CE Workflow

Library Pooling

Cleanup of Library products

Library Construction

Library Quantitation

Cleanup of PCR products
Clonal Amplification/Cluster Generation

Sequence 

Data Analysis

General Workflow for Next Generation Sequencing (NGS)

DNA Extraction

DNA Quantitation

Targeted DNA Amplification Detection by CE Data Analysis 

CE Workflow

Library Pooling

Cleanup of Library products

Library Construction

Library Quantitation

Cleanup of PCR products

Targeted sequencing of STR markers relies on the PCR-amplification process
What is Library Construction ?
STR typing using CE

Library preparation is essential for successful sequencing

D 1 8 S 5 1 CE

D 1 8 S 5 1

T H 0 1

T H 0 1

Dye-labeled STR amplicons

Adapters

T H 0 1

D 1 8 S 5 1

D 1 8 S 5 1

T H 0 1

Adapter STR amplicon Adapter

D 1 8 S 5 1

D 1 8 S 5 1

T H 0 1

T H 0 1

STR amplicons

The aim of library preparation is to flank amplified STR products with adapters 

on both ends

DNA Libraries
Data Analysis using CE technology vs. NGS technology
Data Analysis by CE

❖Separation by size

❖Length variation: 15

❖Peak Height in RFU

RFU
Data Analysis by CE

❖Separation by size

❖Length variation: 15

❖Peak Height in RFU

RFU

+ Sequence variation

[TAGA]11 [CAGA]3 TAGA

[TAGA]10 [CAGA]4 TAGA

Depth of coverage

❖Separation by size

✓ Length variation: 15

❖Peak Height in RFU

Data Analysis by NGS

0

500

1000

1500

2000

2500

3000

3500

15 a 15 b

Coverage

vWA

Bioinformatics pipeline
Why implement NGS if you can accomplish DNA typing by CE?
▪ Examine one marker type at a time in one sample 

Current Markers used in Forensic Genetics

Autosomal 

STRs

Y-STRs

X-STRs

SNPs

Control region 

of mtDNA

CE

NGS Sequencing Application and Markers

NGS

❖ Autosomal STRs 

❖ Y STRs 

❖ X STRs 

❖ Autosomal SNPs

❖ Y-SNPs

Ancestry SNPs

Phenotype SNPs 

Pharmacogenetics 

SNPs

Microhaplotypes

Whole mt-Genome

Microbial 

Identification

DNA methylation

mRNA profiling

▪ Multiplex samples

▪ Multiplex markers

▪ Distinguish between alleles identical by length but 

different in sequence content
Forensic labs are moving from threshold based systems towards fully continuous 

and probabilistic DNA interpretation systems
PGs are based on models that describe 

the behavior of DNA profiles by:

Understanding allele 

and stutter peak height variability

Recognizing the limitations

of the interpretation methodology 

Studying signal saturation

Validating STR multiplexes

Determining allele drop-out 

and drop-in

Current considerations of the CE probabilistic genotyping (PG) systems
What do we need to understand to establish STR NGS 

interpretation systems?
We need to understand and analyze the STR NGS sequence data

Characterization of SingleSource NGS DNA Profiles

Noise sequences 

Stutter sequences Zygosity

Heterozygote sequence balance

Allele drop-in/drop-out

Validate NGS STR multiplexes Effect of sample multiplexing
CE and NGS sensitivity experimental design
CE Workflow

Detection by CE

RFU = 1

DNA Extraction

3 samples

500 pg, 250 pg, 125 pg, 60 pg, 30 pg and 15 pg

PowerPlex PowerPlex 46GY prototype Fusion

Coverage ≥ 1 

Library Construction

TruSeq

Bead-based PCR Cleanup 

0.7X Bead-based Library Cleanup

Sequence 

STRait-Razor 

CE and NGS sensitivity experimental design

➢ Three unique samples selected

➢ Run in triplicate

▪ Three unique amplifications of the serial dilutions

➢ Dilution points

▪ 0.5 ng, 0.25 ng, 0.125 ng, 0.0625 ng, 0.03 ng, and 0.015 ng

Stochastic effects
Noise Thresholds for CE Data
Analytical Threshold Most Commonly Determined by:

Average RFU signal STDEV of the signal 

• k = Numerical factor (e.g. k=3)

AT = μ + 3*σ 

AT = μ + 10*σ 

J. Bregu, D. Conklin, E. Coronado, M. Terrill, R.W. Cotton, C.M. Grgicak, Analytical thresholds and sensitivity: establishing RFU thresholds for forensic DNA 

analysis, Journal of forensic sciences 58(1) (2013) 120-9.
AT level is set at 1.5% of total locus coverage

Removal of general noise using thresholds created by fitting the 

distribution of general noise sequences.

AT = c * (Maxnoise- Minnoise)

Noise Thresholds for NGS Data
Characterization of sequences in STR profiles generated on MiSeq platform 

using the PowerSeq 46GY prototype kit
Total Type of Sequences = 646 

D21S11 (29, 31) Locus Coverage = 6949

Coverage

27

28 29 30 31

We grouped the generated sequences intro three categories:

A = True known allele sequences

S1 S1

S1 = Back stutter of the longest uninterrupted stretch of the basic repeat motifs within an allelic sequence

S2

S2 = Back stutter sequences not attributed to S1

N = Noise sequences

S2 N N N N
Distribution of Known Allele, Stutter, and Noise Sequences

TruSeq-15 pg

A N S1 S2

Percent Coverage

TruSeq-125 pg

Percent Coverage

A N S1 S2

CE-125 pg

Percent Coverage

A N S1 + S2

Percent Coverage

CE-15 pg

A N S1 + S2

As expected, improved discrimination between known alleles (A) and the remainder of the sequences 

(N, S1, and S2) is observed as the amount of DNA template increases.
30

Observed Sequences and their coverage at a heterozygote D21S11 Locus 

D21S11 (29, 31) Locus Coverage = 6949

Y-axis in log scale 

Coverage

29

278 types

31

257 types 

28

50 types

30

39types

27

8 types

AT = μ + 10*σ

AT = % Coverage * Locus Coverage

AT = Range of Noise
At a percent coverage of 15%:

▪ 361 sequences are called 

▪ 350 can be attributed to A

▪ 8 can be attributed to N

▪ 3 can be attributed to S1

▪ 0 can be attributed to S2

Evaluating the tradeoff between the allelic (true positives), stutter, and noise sequences (false

positives)

TruSeq-15 pg

A N S1 S2

Percent Coverage

15 %

Percent Coverage

CE-15 pg

A N S1 + S2

15 %

At a percent coverage of 15%:

▪ 363 peaks are called 

▪ 352 can be attributed to A

▪ 21 can be attributed to N

▪ 0 can be attributed to S
TruSeq-125 pg

Percent Coverage

A N S1 S2

15 %

At a percent coverage of 15%:

▪ 411 sequences are called 

▪ 411 can be attributed to A

▪ 0 can be attributed to N

▪ 0 can be attributed to S1

▪ 0 can be attributed to S2

CE-125 pg

Percent Coverage

A N S1 + S2

15 %

At a percent coverage of 15%:

▪ 411 peaks are called 

▪ 411 can be attributed to A

▪ 0 can be attributed to N

▪ 0 can be attributed to S

Evaluating the tradeoff between the allelic (true positives), stutter, and noise sequences

(false positives)

A value of 15 % is ONLY used for illustrative purposes and not as a recommended threshold. Each lab should 

perform sensitivity experiments and establish a threshold for interpretational purposes.
Summary

▪ Understanding the behavior of STR NGS profiles can help in statistical modeling and

probability distributions needed for establishing an STR NGS interpretation system.

▪ Future work will focus on analyzing more single source and mixture samples.

https://strbase.nist.gov/pub_pres/Sarah-ISHI2018-Poster-SR-final_pmv.pdf

Presentation will be available for download from STRBase: 

http://strbase.nist.gov/NISTpub.htm#Presentations
Acknowledgement 

NIST

Pete Vallone

Hari Iyer (Statistical Design, Analysis, and Modeling Group)

Lisa Borsuk 

Becky Steffen

Erica Romsos

Katherine Gettings

Kevin Kiesler 

Margaret Kline

Megan Cleveland

Funding

NIST Special Programs Office: Forensic DNA

FBI Biometrics Center of Excellence: Forensic DNA

Typing as a Biometric tool.

All work presented has been reviewed and approved by

the NIST Human Subjects Protections Office.

Promega

Doug Storts

Spencer Hermanson Contact: sarah.riman@nist.gov