Reducing noise and stutter in short tandem repeat loci with unique molecular identifiers

Abstract

Unique molecular identifiers (UMIs) are a promising approach to contend with errors generated during PCR and massively parallel sequencing (MPS). With UMI technology, random molecular barcodes are ligated to template DNA molecules prior to PCR, allowing PCR and sequencing error to be tracked and corrected bioinformatically. UMIs have the potential to be particularly informative for the interpretation of short tandem repeats (STRs). Traditional MPS approaches may simply lead to the observation of alleles that are consistent with the hypotheses of stutter, while with UMIs stutter products bioinformatically may be re-associated with their parental alleles and subsequently removed. Herein, a bioinformatics pipeline named strumi is described that is designed for the analysis of STRs that are tagged with UMIs. Unlike other tools, strumi is an alignment-free machine learning driven algorithm that clusters individual MPS reads into UMI families, infers consensus super-reads that represent each family and provides an estimate the resulting haplotype’s accuracy. Super-reads, in turn, approximate independent measurements not of the PCR products, but of the original template molecules, both in terms of quantity and sequence identity. Provisional assessments show that naïve threshold-based approaches generate super-reads that are accurate (∼97 % haplotype accuracy, compared to ∼78 % when UMIs are not used), and the application of a more nuanced machine learning approach increases the accuracy to ∼99.5 % depending on the level of certainty desired. With these features, UMIs may greatly simplify probabilistic genotyping systems and reduce uncertainty. However, the ability to interpret alleles at trace levels also permits the interpretation, characterization and quantification of contamination as well as somatic variation (including somatic stutter), which may present newfound challenges.