High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

Xiangpei Zeng; Jonathan L King; Monika Stoljarova; David H Warshauer; Bobby L LaRue; Antti Sajantila; Jaynish Patel; Douglas R Storts; Bruce Budowle

doi:10.1016/j.fsigen.2014.11.022

High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

Forensic Sci Int Genet. 2015 May:16:38-47. doi: 10.1016/j.fsigen.2014.11.022. Epub 2014 Dec 3.

Authors

Xiangpei Zeng¹, Jonathan L King¹, Monika Stoljarova¹, David H Warshauer¹, Bobby L LaRue¹, Antti Sajantila², Jaynish Patel³, Douglas R Storts³, Bruce Budowle⁴

Affiliations

¹ Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
² Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA; Department of Forensic Medicine, Hjelt Institute, University of Helsinki, P.O. Box 40, 00014 Helsinki, Finland.
³ Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.
⁴ Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA; Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia. Electronic address: Bruce.Budowle@unthsc.edu.

PMID: 25528025
DOI: 10.1016/j.fsigen.2014.11.022

Abstract

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

Keywords: Illumina MiSeq; Massively parallel sequencing (MPS); Short tandem repeat (STR).

Publication types

Comparative Study
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

DNA / analysis
DNA / genetics
DNA Fingerprinting / methods*
Electrophoresis, Capillary / methods
Forensic Genetics / methods*
Gene Frequency
High-Throughput Nucleotide Sequencing / methods*
Humans
Microsatellite Repeats*
Multiplex Polymerase Chain Reaction / methods*
Specimen Handling

Substances

DNA