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URL: https://dx.doi.org/10.17504/protocols.io.9i7h4hn

Authors: Gregory Harhay

Summary: The virulence and pathogenicity of bacterial pathogens are related to their adaptability to changing environments. One process enabling adaptation is based on minor changes in genome sequence, as small as a few base pairs, within segments of genome called simple sequence repeats (SSRs) that consist of multiple copies of a short sequence (from one to several nucleotides), repeated in series.  SSRs are found in eukaryotes as well as prokaryotes, and variation in them occurs at frequencies up to a million-fold higher than the average bacterial mutation rate through a process of slipped stranded mispairing (SSM) by DNA polymerase during replication. The characterization of SSR length by standard sequencing methods is complicated by the appearance of length variation introduced during the sequencing process that does not accurately quantify lower-abundance repeat number variants in a population. Here we report a computational approach to correct for process-induced artifacts, validated for tetranucleotide repeats by use of synthetic constructs of fixed, known length. We apply this method to a laboratory culture ofHistophilus somni, prepared from a single colony, and demonstrate that the culture consists of populations of distinct sequence phase and read length variants at individual tetranucleotide SSR 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 CCS (circular consensus sequence) wiith repetitve sequence to closed genomes are guaranted to map to a single locus if sufficent unique flanking sequence is used to confim the unique mapping. Consequently, long CCS with high base quality are the most desirable input into this workflow.

Affiliations: United States Department of Agriculture

Version: 5

Publication Date: 2019

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