Variant Allele Characterization in STR Markers Using Next Generation Sequencing

Published

May 29, 2026

Author(s)

Lauren Mullen, Carolyn Steffen, Katherine Gettings, Kevin Kiesler, Peter Vallone

Abstract

Traditionally, Sanger sequencing was used to characterize reference materials and confirm discordant allele calls from different STR typing kits at the National Institute of Standards and Technology (NIST). Sequencing can also identify genomic variations within polymerase chain reaction (PCR) amplicons containing STRs, particularly variants that result in null alleles and alleles that do not migrate within allele sizing bins provided by kit manufacturers. However, Sanger methods are low-throughput, time- and labor-intensive, and require additional procedures for analysis of heterozygous alleles. To address these limitations, a quicker, more straightforward protocol that uses next-generation sequencing (NGS) was developed. By applying NGS technology to forensic samples containing variant alleles, additional information can be obtained about their molecular basis, and this information can be published and shared across the forensic community. The development of this protocol can increase awareness and encourage the integration of NGS technology into forensic laboratories to improve forensic DNA typing for human identification. This research provides the criteria used to individually sequence thirty-five autosomal STR loci, with PCR primer locations chosen to increase amplicon length and maximize the likelihood of detecting variants in the flanking region. The list of targeted sequences, their primers, and the chromosomal coordinates is also included.

Citation

MDPI Genes

Volume

17

Issue

6

Pub Type

Journals

Download Paper

https://doi.org/10.3390/genes17060617

Local Download

Keywords

Variant allele, Null allele, Off-ladder allele, Next generation sequencing

Citation

Mullen, L. , Steffen, C. , Gettings, K. , Kiesler, K. and Vallone, P. (2026), Variant Allele Characterization in STR Markers Using Next Generation Sequencing, MDPI Genes, [online], https://doi.org/10.3390/genes17060617, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=961966 (Accessed June 18, 2026)

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