Applied 

Genetics

STR Loci and 

Multiplex Kits

Becky Hill

Research Biologist, Applied Genetics Group

Forensics@NIST 2012 Meeting

Gaithersburg, MD

November 28, 2012
Applied 

Genetics Outline of Topics to Discuss

• Brief background on STR loci and kits

– What are they and why are they important?

• The role of NIST in STR typing

– NIST unrelated 1036 U.S. population samples

– Concordance testing

• New STR multiplex kits available

– GlobalFiler (Life Technologies)

– PowerPlex Fusion (Promega)

– PowerPlex Y23 (Promega)
Applied 

Genetics

Background on STR Loci and Kits
Steps in DNA Analysis

DNA 

Extraction

Multiplex PCR Amplification

Male: 13,14-15,16-12,13-10,13-15,16

Interpretation of Results

Sample Collection & 

Storage

Blood Stain Buccal swab

DNA 

Quantitation

Slot Blot

1 ng

0.3 ng

1 ng

1 ng

0.7 ng

0.5 ng

0.5 ng

No DNA

Usually 1-2 day process (a minimum of ~5 hours)

If a match occurs, comparison of DNA 

profile to population allele 

frequencies to generate a case report 

with probability of a random match to 

an unrelated individual

STR Typing

DNA separation and sizing

Technology Biology

Genetics

DNA 

Database 

Search 

Collection

Extraction

Quantitation

STR Typing

Interpretation 

of Results

Database Storage 

& Searching

Specimen Storage

Multiplex PCR

Calculation of 

Match Probability

Steps Involved
Short Tandem Repeat (STR) Markers

TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAGACAGGTGGATAGATA

GATAGATAGATAGATAGATAGATAGATAGATAGATAGATATCATTGAAAGACAAA

ACAGAGATGGATGATAGATACATGCTTACAGATGCACAC

= 12 GATA repeats (“12” is all that is reported)

Target region 

(short tandem repeat)

7 repeats

8 repeats

9 repeats

10 repeats

11 repeats

12 repeats

13 repeats

The number of consecutive repeat units 

can vary between people

An accordion-like DNA sequence that occurs between genes

The FBI has selected 13 

core STR loci that must be 

run in all DNA tests in 

order to provide a 

common currency with 

DNA profiles
Fluorescent 

dye-labeled 

primer

GATA

3′

5′

3′

5′

(Maternal)

(Paternal)

1 2 3 4 5 6

1 2 3 4 5 6 7 8

STR Repeat Region

forward primer 

hybridization region

reverse primer 

hybridization region

75....80....100....120....140....160....180....200....220....240....260.....

(size in bp) 

RFUs

1000

500

6

139bp

8

147bp

DNA Separation and Detection

Short Tandem Repeat (STR) Typing
Applied 

Genetics

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

13 Core U.S. STR Loci

AMEL

AMEL

Sex-typing

Position of Forensic STR Markers on 

Human Chromosomes

8 STR loci overlap between U.S. and Europe

1997
Applied 

Genetics

Commercially Available STR Kits

Applied Biosystems (18)

• AmpFlSTR Blue (1996)

• AmpFlSTR Green I (1997)

• Profiler (1997)

• Profiler Plus (1997)

• COfiler (1998)

• SGM Plus (1999)

• Identifiler (2001)

• Profiler Plus ID (2001)

• SEfiler (2002)

• Yfiler (2004)

• MiniFiler (2007)

• SEfiler Plus (2007)

• Sinofiler (2008) – China only

• Identifiler Direct (2009)

• NGM (2009)

• Identifiler Plus (2010)

• NGM SElect (2010)

• GlobalFiler (2012)

Promega Corporation (17)

• PowerPlex 1.1 (1997)

• PowerPlex 1.2 (1998)

• PowerPlex 2.1 (1999)

• PowerPlex 16 (2000)

• PowerPlex ES (2002)

• PowerPlex Y (2003)

• PowerPlex S5 (2007)

• PowerPlex 16 HS (2009)

• PowerPlex ESX 16 (2009)

• PowerPlex ESX 17 (2009)

• PowerPlex ESI 16 (2009)

• PowerPlex ESI 17 (2009)

• PowerPlex CS7 (2009)

• PowerPlex 18D (2011)

• PowerPlex Y23 (2012)

• PowerPlex 21 (2012)

• PowerPlex Fusion (2012)

Qiagen (10) kits in 2010

Primarily selling kits in Europe

Due to patent restrictions 

cannot sell in U.S.

Investigator kits

• ESSplex

• ESSplex SE

• Decaplex SE

• IDplex

• Nonaplex ESS

• Hexaplex ESS

• HDplex

• Triplex AFS QS

• Triplex DSF

• Argus X-12

>1/3 of all STR kits were released in the last four years
Impact of DNA Amount into Multiplex PCR Reaction

DNA amount

(log scale)

0.5 ng

-A

+A

Too much DNA

 Off-scale peaks

 Split peaks (+/-A)

 Locus-to-locus imbalance

100 ng

10 ng

1 ng

0.1 ng

0.01 ng

2.0 ng

Too little DNA

 Heterozygote peak imbalance

 Allele drop-out

 Locus-to-locus imbalance

Stochastic effects when amplifying low 

levels of DNA can produce allele dropout

STR Kits Work Best in This Range

High levels of DNA create interpretation 

challenges (more artifacts to review)

Well-balanced STR multiplex

We generally aim for 0.5-2 ng

100 pg 

template

5 pg 

template
Applied 

Genetics

NIST U.S. Population Samples
NIST Standard Sample Sets

• NIST U.S. population samples

– 260 African American, 260 Caucasian, 140 Hispanic, 3 Asian

• U.S. father/son paired samples

– ~100 fathers/100 sons for each group: 200 African American, 

200 Caucasian, 200 Hispanic, 200 Asian

• NIST SRM 2391b, PCR-based DNA Profiling Standard 

(highly characterized)

– 10 genomic DNA samples, 2 cell line samples

– Includes 9947A and 9948

• NIST SRM 2391c, PCR-based DNA Profiling Standard

– 4 genomic DNA (one mixture)

– 2 cell lines (903 and FTA paper)

Stock tubes

extracted genomic 

DNA

>1450 total samples

http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm
Applied 

Genetics NIST 1036 U.S. Population Samples

• 1032 males + 4 females

– 361 Caucasians (2 female)

– 342 African Americans (1 female)

– 236 Hispanics

– 97 Asians (1 female)

• Anonymous donors with self-identified ancestry

– Interstate Blood Bank (Memphis, TN) – obtained in 2002

– Millennium Biotech, Inc. (Ft. Lauderdale, FL) – obtained in 2001

– DNA Diagnostics Center (Fairfield, OH) – obtained in 2007

• Complete profiles with 29 autosomal STRs + PowerPlex Y23

– Examined with multiple kits and in-house primer sets enabling concordance

• Additional DNA results available on subsets of these samples

– mtDNA control region/whole genome (AFDIL)

– >100 SNPs (AIMs), 68 InDel markers, X-STRs (AFDIL)

– NIST assays: miniSTRs, 26plex, >100 Y-STRs, 50 Y-SNPs

Unrelated samples

All known or potential related individuals 

(based on autosomal & lineage marker testing) 

have been removed from the 1036 data set 

(e.g., only sons were used from father-son samples)

Data available on STRBase: http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm
Applied 

Genetics Benefits of NIST 1036 Data Set

• Elimination of potential null alleles due to primer 

binding site mutations through extensive concordance 

testing performed with different PCR primer sets from all 

available commercial STR kits

• Ancestry testing performed on DNA samples with 

autosomal SNPs, Y-SNPs, and mtDNA sequencing to 

verify self-declared ancestry categorization

• Related individuals removed based on Y-STR and 

mtDNA results

• Full characterization of all commercial STR loci based 

on population statistics
Characterizing New STR Loci

Main Points:

• In April 2011, the FBI announced plans to expand the core 

loci for the U.S. beyond the current 13 CODIS STRs

• Our group is collecting U.S. population data on new loci 

and characterizing them to aid understanding of various 

marker combinations

• We are collecting all available information from the 

literature on the 29 commonly used autosomal STR loci 

and 23 Y-STR loci

Presentations/Publications:

• Hill et al (2011) FSI Genetics 5(4): 269-275

• Hares (2012) Expanding the U.S. core loci... FSI Genetics 6(1): e52-e54

• Butler & Hill (2012) Forensic Sci Rev 24(1): 15-26

• Hill et al (2012) poster at ISHI: 

http://www.cstl.nist.gov/biotech/strbase/pub_pres/Hill-ISHI2012-STRloci.pdf
Applied 

Genetics NIST U.S. Population Data

• The data from our 1036 U.S. population samples 

is now available on STRBase:

• A summary of the NIST 1036 data set was 

recently published in Profiles in DNA for 

autosomal and YSTR loci 

• Population data have been submitted to 

FSI: Genetics for publication

– 29 autosomal STR loci (Hill et al)

– 23 Y-STR loci (Coble et al)

http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm
Applied 

Genetics

Concordance Evaluation of STR Kits
Working with Forensic Commercial Companies

• Work primarily with 3 forensic commercial 

companies

– Applied Biosystems – Foster City, CA 

– Promega – Madison, WI

– Qiagen - Germantown, MD (parent company in Hilden, 

Germany)

• What is provided to NIST

– Prototype DNA kits for evaluation with NIST standard 

U.S. samples

• What we provide to the companies

– Complete data evaluation and publications of findings 

with their approval 
Importance of Data Comparisons 

Between DNA Kits

• There are a variety of commercial DNA kits with different 

configurations of markers

– Differences in profiles are rare, but can and do occur

• Discordant results can impact DNA databases

– Currently ~11.5 million profiles in the national database

– Information sharing occurs between state and local databases

– If there are differences between kits, this can be detrimental (could 

lead to false negatives)

• Concordance with NIST reference materials is valuable for 

proper calibrations of all kits used

– All forensic labs are required to test NIST SRM 2391c per FBI QAS

– Important to test with all new DNA kits to determine and characterize 

any differences
STR Kit Concordance Studies

• Examined NIST samples 

across >20 STR kits and inhouse assays covering 29 

autosomal STR loci

• 99.90% concordance 

observed to-date

– 1,225 total differences due to 

primer binding site mutations 

from 1,176,994 allele 

comparisons (as of Nov 2012)

• Information provided back 

to kit developers to redesign 

primers or add extra ones –

often prior to kit release

D18S51 Comparisons

D18S51 null allele with the NGM SElect kit as compared to 

the ESSplex SE kit, PowerPlex ESX 17 and ESI 17 systems

Kits are kindly provided by Applied 

Biosystems, Promega, and Qiagen for 

concordance testing performed at NIST
Benefits to the Forensic Community

• With NIST input and testing, companies can fix their 

kits before they are released and allow them get to 

the market faster with fewer issues

• The market share for these DNA analysis systems 

(instrumentation and reagents) is in the $150-200 M per 

year

– NIST has a direct impact by helping to improve the technology

• NIST has a neutral and confidential approach to working 

with different commercial companies simultaneously

• NIST has access to a useful set of standard U.S. 

population samples for these kit evaluations

• NIST publications in peer-reviewed journals and 

presentations in domestic and international conferences 

and webinars
Applied 

Genetics

New STR Multiplex Kits

Recently Launched
Promega PowerPlex FUSION (5-dye – CC5 internal lane standard 500)

FGA

A

vWA

D18S51

D8S1179 D19S433

D21S11

D3S1358

TH01

D16S539 D2S1338

D22S1045

D2S441 Penta E

100 bp 200 bp 300 bp 400 bp

D1S1656

D12S391

D10S1248

D5S818

D13S317

D7S820

CSF1PO

TPOX DYS391

24plex

FL

JOE

TMR-ET

CXR-ET

Penta D

Life Technologies/Applied Biosystems GlobalFiler (6-dye – LIZ600 size standard)

FGA

A

vWA

D18S51

D19S433

D8S1179 D21S11

D3S1358

TH01

D16S539

D2S1338

D22S1045

D2S441

SE33

100 bp 200 bp 300 bp 400 bp

D10S1248 D1S1656 D12S391

D5S818 D13S317 D7S820

CSF1PO

Y DYS391

TPOX

24plex

6-FAM

VIC

NED

TAZ

SID

* *

*

* * * *

8 new degenerate primers added

Expanded D2, D8 ladder alleles

343 alleles & 245 virtual bins

Rapid 2-step PCR

STR Kit Layouts by Dye Label and PCR Product Size
Applied Biosystems GlobalFiler

Promega PowerPlex Fusion

Both kits 

were 

released 

in Sept 

2012
DYS549 DYS533 DYS438 DYS437

DYS570 DYS635 DYS390 DYS439 DYS392

DYS391 DYS481

DYS576 DYS389I DYS448 DYS389II DYS19

DYS643

DYS393 DYS458 DYS385a_b DYS456 Y-GATA-H4

PowerPlex Y23 Allelic Ladders
Applied 

Genetics Summary

• NIST has a set of 1036 U.S. population samples 

that have been used to fully characterize 29 

autosomal STR loci and 23 Y-STR loci

• NIST plays an important role in concordance 

testing to aid the community

– Several null alleles have been fixed before the final 

release of new STR multiplex kits

• Commercial companies are continuing to 

release larger STR multiplexes to meet the 

needs of the forensic community
Applied 

Genetics Acknowledgments

Contact Info: 

becky.hill@nist.gov

301-975-4275

NIST Team for This Work

Funding from the 

National Institute of 

Justice (NIJ) through 

NIST Office of Law 

Enforcement Standards

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 those of the presenters and do not necessarily represent the official position of the National Institute of Standards and 

Technology or the U.S. Department of Justice.

John Butler Dave Duewer Margaret Kline Mike Coble

A special thanks to 

Applied Biosystems, 

Promega, and Qiagen

for providing the kits 

used in this study
Applied 

Genetics Thank you for your attention!