Computational Steps for Analyzing Deep Single Molecule Sequencing Reads of Simple Sequence Repeats - Genomic SSRs v2
Authors: P Harhay Gregory 
Published: December 2018

The adaptability of bacterial pathogens is a crucial attribute related to their virulence and pathogenicity. A characteristic of bacterial adaptation is an ability to rapidly and reversibly reprogram gene expression in response to environmental cues. One process that has evolved in bacteria involves relatively minor changes in genome sequence corresponding to simple sequence repeats (SSR). These are segments of genome made up of multiple copies of a short sequence (from one to several nucleotides), repeated in series, although their potential biological role(s) in eukaryotes (Toth 2000) or prokaryotes (Moxon 2006) is not fully understood. SSR length variation has been shown to affect phenotype in multiple bacterial species (van der Woude 2004) and occurs through slip-stranded mispairing (SSM) of DNA at a rate of up to a million-fold greater than the average bacterial mutation rate (Moxon 2006). This hypermutability 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 report a process that utilizes single-molecule real-time (SMRT) sequencing to analyze SSRs in bacterial colonies or cultures that discriminates between sequencing artifact and biological signal. Using this method, we demonstrate that a laboratory culture of Histophilus somni, prepared from a single, presumably clonal colony, consists of a population of distinct sequence phase and read length variant profiles at individual SSR loci. These results suggest SSM-mediated phase variation in bacterial populations inherently contain quantifiable subpopulations, each with their own environmental fitness, with some rapidly expanding upon environmental perturbation that accounts for their observed adaptability. We show that specific SSR regions in Histophilus somni are not static structures well-represented by a single consensus sequence, but rather dynamic structures with length variation differences in multiples of complete repeat units, having characteristic SSM profiles and sequence phase. Immediate applications of this method include direct monitoring of SSM-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 polymerases and regulatory biomolecules with variations in SSR repeat unit sequence, repeat unit sequence phase, repeat unit length, and hypermutable total SSR length at individual loci. 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.