Sequence-based analysis of stutter at STR loci: Characterization and utility

The development of next generation sequencing (NGS) technologies creates the potential for changing the method by which the forensic science community genotypes short tandem repeat (STR) loci. While the capabilities of NGS are promising, moving from current capillary electrophoresis (CE) methods would require new guidelines to be established and a new understanding of artifacts that may arise with the use of NGS. Stutter has been well characterized for CE technologies; however, NGS workflows may use different polymerases and amplification approaches, which could alter the appearance of this artifact. Stutter is most commonly seen in the n-4 position in CE data, but may be observed more rarely in the n+4 and n-8 positions. NGS data frequently contains detectable sequences consistent with stutter at the n+4 and n-8 positions, and may even contain stutter at the n-12 position for some loci. It is possible that these alternate types of stutter events occur at similar levels in CE workflows and go undetected due to the analytical threshold employed or because the artifacts do not exceed the background noise. Comparing stutter events in NGS data to what has been observed by CE will improve our understanding of the effects of library preparation and sequencing. Characterizing stutter events by sequence will contribute to the development of guidelines and facilitate implementation of NGS technology. Further, determining stutter ratios for each isoallele would allow for individual sequence thresholds to be set, which could then be used to improve mixture interpretation models.