SiMSen-Seq STR sequencing across five laboratories enables lowered noise thresholds and improved allele calling

Unique molecular identifiers (UMI) can be used in forensic STR sequencing to reduce the level of analytical artifacts. Here, we perform an interlaboratory study across five independent sites where the previously developed UMI-based SiMSen-Seq STR assay is applied at each laboratory to both single-source and mixed samples. The assay showed consistent results between laboratories and more than 90% of the expected alleles were detected with 31 pg DNA of template. The assay tolerated ten times higher PCR inhibitor concentrations compared to an established commercial STR sequencing method. The combined results were used to determine stutter and noise thresholds, which were applied for allele calling, allowing an estimation of the sensitivity for minor contributor alleles in mixtures. We found that the SiMSen-Seq STR method is robust across laboratories and different types of PCR and sequencing equipment and that it allows for calling of alleles from contributors of smaller proportions compared to currently commercially available non-UMI STR sequencing methods.