Computational Steps for Analyzing Deep Single Molecule Sequencing Reads of Simple Sequence Repeats - Genomic SSRs v1
Authors: P Harhay Gregory 
Published: November 2018

Simple sequence repeats (SSR) are segments of genome sequence made up of multiple copies of a short sequence (from one to several nucleotides), repeated in series. SSRs are present in prokaryotes(Moxon 2006)and eukaryotes(Toth 2000)however, their biological roles are not fully understood. In some bacterial genomes, SSR length variation has been shown to affect phenotype(van der Woude 2004). SSR length variation occurs through slip-stranded mispairing (SSM) of DNA at a rate of up to a million-fold greater than the average bacterial mutation rate and is therefore termed hypermutable(Moxon 2006). This mode of mutation has been observed in both gram-positive(Danne 2014)and gram-negative bacteria(Inzana 2013)and shown to affect host adaptation, virulence, and pathogenicity. Here we use single-molecule real-time sequencing and a dedicated data processing pipeline to demonstrate that a laboratory culture ofHistophilussomni, prepared from a single, presumably clonal colony, consists of a population ofdistinctsequence phase and read length variant profiles at individual SSR loci. To discriminate between sequencing artifact and biological signal, we employed chemically synthesized control duplexes that we sequenced to create a dataset to tune our computational approach to account for artifacts.Using these control duplexes, we observed low levels of SSM for thePacBio polymerase used in SMRT sequencing.To the best of our knowledge, we present the first protocol for the observation of SSR length variabilityat the single DNA molecule level that accounts for sequencer SSM artifact. Our results demonstrate a method to detect and quantify SSR length heterogeneity in a mixed population of DNA molecules and show that specific SSR regions inHistophilus somniare not static structures well-represented by a single consensus sequence, but rather dynamic structures with length variation differences in multiples of complete repeat unit with characteristic SSM profiles and sequence phase. Immediate applications of this method include direct monitoring of SSR-mediated phase variable gene switching and the development of genome assemblies and annotation that more accurately reflect the sequence diversity of extant genomes. This method may be more broadly applied to study the potentially species-specific interactions of DNA and RNA polymerases and regulatory biomolecules on the one hand, with variations in SSR repeat unit sequence, repeat unit sequence phase, repeat unit length, and hypermutable total length on the other. Input requirements: Closed Genome - It is recommended that only organisms with closed genomes be the subject of the analyses described here. Mapping repetitive reads to to contigs of non-closed genomes may map to multiple locations, complicating tha analysis. Mapping reads wiith reptitve sequence to closed genomes are guaranted to map to a single locus if sufficent unique flanking sequence is used to confim the unique mapping. For this unique mapping to be effective, each read describiing the region of interest must span not only the SSR, but the unique flanking sequence. Consequently, long reads with high base quality are the most desirable input into this workflow.