STR 26plex Assay

[Information on Loci] [PCR Primer Sequences] [Example Data] [Bins and Panels] [Protocols] [Additional Information]

Information on Loci:

Hill, C.R., Kline, M.C., Coble, M.D., Butler, J.M. (2008) Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples. J. Forensic Sci. 53(1):73-80.

 

An 8plex assay has also been developed with a subset of these loci...[link]

PCR Primer Sequences:

Hill, C.R., Butler, J.M., Vallone, P.M. (2008) A 26plex autosomal STR assay to aid human identity testing. J. Forensic Sci., in press.

Locus Name

Forward Dye Label

Primer Sequence (5' - 3')

Primer Concentration, µM

GenBank  accession

D1GATA113

PET

F - acattaagcacatgcctctttgt

1

Z97987

 

 

R - Gatgaactcattggcaaaagga

1

 

D1S1627

NED

F - catgaggtttgcaaatactatcttaac

2

AC093119

 

 

R - Gttttaattttctccaaatctcca

2

 

D1S1677

6FAM

F - gcagtcagcttgattgatcc

3

AL513307

 

 

R - GTTTCTTagaatgcaatagcaaatatcagaatg

3

 

D2S441

NED

F - caaaaggctgtaacaagggcta

1

AC079112

 

 

R - Gttcactctccttcccaaatgtt

1

 

D2S1776

VIC

F - ttacctgtgagtatgtgtgcgta

1.5

AC009475

 

 

R - ggtgctaggtgtgctcagga

1.5

 

D3S3053

VIC

F - tgacacaaatggaccaagaca

2

AC069259

 

 

R - GTTTCTTgagagagcccttgaaatagca

2

 

D3S4529

NED

F - cccaaaattacttgagccaat

0.75

AC117452

 

 

R - Gagacaaaatgaagaaacagacag

0.75

 

D4S2364

VIC

F - ctaggagatcatgtgggtatgatt

0.75

AC022317

 

 

R - Gcagtgaataaatgaacgaatgga

0.75

 

D4S2408

NED

F - agctgacatcttaccacatgttc

2

AC110763

 

 

R - Gtgcttggcatatattaagacactgta

2

 

D5S2500

VIC

F - gtttactgataaaccaaatgatgtgc

2

AC008791

 

 

R - Gtaacttaaagggtaaatgtttgcag

2

 

D6S474

6FAM

F - ggttttccaagagatagaccaatta

1.5

AL357514

 

 

R - Gtcctctcataaatccctactcatatc

1.5

 

D6S1017

NED

F - agatgggaacgatgcagaca

2

AL035588

 

 

R - gcataaatggatgggtggat

2

 

D8S1115

PET

F - gcaccatttcacatatcca

6

AC090739

 

 

R - Gcagtctcctaagtagc

6

 

D9S1122

VIC

F - gggtatttcaagataactgtagatagg

0.75

AL161789

 

 

R - Gcttctgaaagcttctagtttacc

0.75

 

D9S2157

NED

F - gatcacgccacggta

5

AL162417

 

 

R - Gttctccatttcaaagatcat

5

 

D10S1248

6FAM

F - cagtaaaaagcaaacctgagca

1

AL391869

 

 

R - gcttggcaaagagcagatg

1

 

D10S1435

VIC

F - cacgttgggtttcctgactt

1

AL354747

 

 

R - Gcccagctacttgggatgcta

1

 

D11S4463

6FAM

F - ctgtcccaaggctgagtgtt

3

AP002806

 

 

R - GTTTCTTcgcagggcaaataaaaagaa

3

 

D12ATA63

6FAM

F - aggtggcagtgagctgtaac

1

AC009771

 

 

R - GTTtcttggattttgagggccta

1

 

D14S14343

PET

F - ggctctgatttccaccactg

2

AL121612

 

 

R - Gcaactcttggaagcccagtc

2

 

D17S974

NED

F - ggaacacttgagcca

2

AC034303

 

 

R - gtggactggggtaagg

2

 

D17S1301

PET

F - aagatgaaattgccatgtaaaaata

2

AC016888

 

 

R - Gtgtgtataacaaaattcctatgatgg

2

 

D18S853

PET

F - acatatataatgtgagaaaggaggagt

2

AP005130

 

 

R - Gttaatgggtgcaacacacc

2

 

D20S482

PET

F - ctccattctctcacacccaat

1

AL121781

 

 

R - Gcacttctggcttttctggttc

1

 

D20S1082

6FAM

F - acatgtatcccagaacttaaagtaaac

1

AL158015

 

 

R - Gcagaagggaaaattgaagctg

1

 

D22S1045

6FAM

F - ccctgtcctagccttcttatagc

1

AL022314

 

 

R - Gctgtgcccaagttgagagaa

1

 

Amelogenin

PET

F - ccctttgaagtggtaccagagca

2

M55418, M55419

 

 

R - gcatgcctaatattttcagggaata

2

 

 

Example Data (1ng DNA, 30 cycles):

 Click to download Bins and Panels for GeneMapperID v3.2

Results with Standard Samples (SRM 2391b components including 9947A, 9948 and K562, ABI 007):

http://www.cstl.nist.gov/biotech/strbase/miniSTR/miniSTR_NC_loci_types.htm

Protocols

26plex assay

 

Additional Information 
 

 

Examples of Artifacts Observed During Multiplex Testing Leading to Primer Redesign

Examples of non-specific products, artifacts, and incomplete adenylation that occurred with three loci (D3S3053, D17S974, D20S1082, respectively) indicating that the primer sets were incompatible and therefore candidates for redesign:

 

An example sequence map in Lasergene software of D3S3053. 

All arrows pointing to the right represent forward primers and the arrows pointing to the left are reverse primers.  The box in the center is the repeat motif and all of the theoretical PCR amplicon “amp” sizes are listed between the primers.  The arrows closest to the repeat box are the miniSTR primers, the arrows labeled with “Seq” are the sequencing primers, the arrows labeled “MMP” are the first set of multiplex primers, and the arrows labeled “MMP new” are the second set of redesigned multiplex primers.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Representative Sensitivity Study data:

 Lowest DNA template amounts for each cycling: 28, 30, and 32 cycles

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Concordance Studies

A modified version of the multiplex (with 22 autosomal loci and amelogenin – 23plex) was used in concordance evaluations with the miniSTR data to determine the presence of null alleles, a mutation rate study with father/son sample pairs to examine mutation rates with these primer sets, and an extended family study to determine how extra autosomal loci affect close family relationship forensic testing. 

 Concordance Study info:

 The use of non-overlapping primers for the majority of the loci permits the detection of allelic drop out.  About half of the dropout is from 23plex primers indicating that the flanking regions near STR repeat do not appear to have a higher level of mutation.

Two examples of discordance between the 23plex primers and the miniSTR primers.  A. The first illustrates a primer binding site mutation in the 23plex primers.  B. The second illustrates a primer binding site mutation in the miniSTR primers.

Mutation Rates

List of calculated mutation rates for the 23plex and all commercially used markers, including the 13 CODIS loci.

STR System

Mutation Rate

  

TH01

0.01%

  

TPOX

0.01%

  

23plex

0.07%

  

D7S820

0.10%

  

D5S818

0.11%

  

D16S539

0.11%

  

D19S433

0.11%

  

D3S1358

0.12%

  

D2S1338

0.12%

  

D8S1179

0.14%

  

D13S317

0.14%

  

Penta D

0.14%

  

CSF1PO

0.16%

  

Penta E

0.16%

  

VWA

0.17%

  

D21S11

0.19%

  

D18S51

0.22%

  

FGA

0.28%

  

SE33

0.64%

  

 Mutation observed in 22 STR loci examined across 395 father/son pairs previously confirmed via Identifiler and Yfiler testing: 

Ethnicity

Locus

Allele (father)

Allele (child)

African American

D6S474

14,16

17,17

Caucasian

D2S1776

13,13

12,14

Hispanic

D11S4463

14,15

13,16

Asian

D10S1435

11,14

12,13

Asian

D3S4529

13,13

15,15

Asian

D20S482

12,15

13,14

 

Extended Family Samples Study

Schematic of the extended family samples received from DNA Diagnostics Center.  This figure illustrates all of the family relationships that were tested in this study.

 Likelihood Ratio (LR) comparisons of different family relationships between the 15 autosomal loci of Identifiler and the 22 autosomal loci of the 23plex + the 15 loci from Identifiler (37 loci total).

Relationship Examined

15 STRs (Identifiler, ID15)

ID15 + 23plex 22 STRs = 37 loci (A37)

Mother/Child*

0.214

5,200,000

Siblings

477

113,000

Uncle/Nephew

824

247,000

Cousins

0.45

2.25

Grandparents/Grandchildren

0.53

1.42

For more information, see Butler, J.M., Hill, C.R., Decker, A.E., Kline, M.C., Reid, T.M., Vallone, P.M. (2007) New autosomal and Y-chromosome STR loci: characterization and potential uses. Proceedings of the Eighteenth International Symposium on Human Identification. Also available at http://www.promega.com/geneticidproc/ussymp18proc/oralpresentations/Butler.pdf.

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Last Updated: 10/21/2008