Short-read, high-throughput sequencing technology for STR genotyping

Abstract

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples.

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

“…However, as predicted nearly five years ago [10], massively parallel sequencing technologies (MPS) are now finding their way into the forensic arena [8,[11][12][13][14][15][16][17][18][19][20]. These technologies largely remove the greatest existing technical and cost barriers to mtGenome sequencing of low DNA quality and quantity specimens.…”

A novel set of DIP-STR markers for improved analysis of challenging DNA mixtures

“…To date, research into the use of MPS for STR mixture resolution by the forensic community has been relatively limited [11,12]. The large volume of data generated with this technology offers interpretation challenges and the method is not yet financially advantageous.…”

Evaluation of the Ion Torrent™ HID SNP 169-plex: A SNP typing assay developed for human identification by second generation sequencing

“…They provide the possibility to (1) obtain detailed sequence information on the traditional forensic genetic markers, (2) type combinations of markers that cannot be typed in the same assay with the currently used methods, and (3) collect massive amounts of data from either one individual or large amount of data from many individuals simultaneously. SGS of STRs [1][2][3][4][5][6] and mtDNA [7,8] Life Technologies TM developed a SNP typing assay for human identification named the Ion Torrent TM HID SNP panel. An early version of this panel (v0.1) was recently tested by Budowle and co-workers [9].…”