The average number of total raw sequenced reads required to correctly identify the CODIS STR alleles with greater than 99% confidence in each sample was less than one million. In addition, the minimum number of reads mapped to the entire in silico reference required to make the same calls was as few as 18,500. However, the limitations of determining longer alleles of D21S11 and potential allele drop-out from D18S51 was further illustrated with the Monte Carlo Model, as increasing number of reads failed to provide 100% accurate genotyping for these loci using this sample set (Figure 2). Similar results were obtained from submitting subsets (10% and 1%) of total sequence reads to the reference aligner (Supplementary Table S4).These data showed that significantly fewer reads than were initially generated were required to make STR genotyping calls by the NGS approach. Consequently, in order to maximize the number of individuals included within a single sequencing run and thereby reduce the cost of analyzing STRs by this method, multiplexing of samples within a large cohort of individuals is recommended.
Here we have demonstrated the potential of using a widely adopted NGS platform for accurately genotyping the CODIS STR loci. Due to the short length of sequence reads attributed to this platform, sequencing STR loci was previously assumed impractical because of their relatively large repeat size (14). The D21S11 locus was the only problematic STR in this regard, as repeat regions extending beyond the 150 bp maximum read length were not resolved. Paired-end (PE) sequencing with overlapping reads could possibly be beneficial for the Illumina GAIIx platform, potentially generating assembled reads longer than 150 bp. However, with simple repeats, the degree of overlap between the paired ends cannot be determined with confidence. Furthermore, STR analysis would not profit from nonoverlapping PE reads with unsequenced inserts, since the reference alignment method requires a contiguous sequence covering the entire STR repeat and some of the unique flanking regions both up- and downstream of the repeat. A final benefit of using the Illumina NGS platform in STR genotyping analyses is the high level of coverage obtained from one sequencing run. Not only did the large sequence data allow for high probability in accuratelycalling alleles at STR loci, it is also amenable for genotyping a large number of individuals. Based on the sensitivity analysis for determining minimum read lengths necessary for accurately calling alleles for a given STR locus, we estimate that >300 individuals could theoretically be STR-genotyped using eight lanes (one flow cell) of an Illumina GAIIx, thereby providing a significant cost and time savings advantage. Alternatively, the massive sequencing bandwidth could be used to evaluate other forensically relevant genetic features, such as ancestry informative markers or forensic DNA phenotypic markers.
This research was funded by an internal research and development program at Battelle Memorial Institute. PSY and BR were supported by NCI Comprehensive Cancer Center Support Grant P30 CA016058.
The authors declare no competing interests. The authors and their institutions do not specifically endorse any of the third-party products or technologies described in this report.
Address correspondence to Seth A. Faith, Battelle Memorial Institute, 505 King Avenue, Columbus, OH, USA. e-mail: email@example.com
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