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.

Keywords: Bridge PCR; Illumina; SNP; STR; forensic; genotyping; high-throughput sequencing; next-generation sequencing.

Figure 1

Visualization of mapped reads to specific alleles of the TPOX locus for two individual samples (index 2 and index 6) and a 1:1 mixture (index 12).

Figure 2. Probability of correct allele assignment for each CODIS STR locus

For each model, 10,000 draws were randomly obtained from a grid of (i) total reads, (ii) reads aligning to the in silico genome, and (iii) the number of reads mapping to the entire locus. The double x-axis represents a cumulative estimate of the (i) number of total raw sequencing reads and (ii) total number of reads aligning to the full STR in silico genome.