Steps to Create FASTQ of CCS Overlapping Control SSR - CCS ROI v6

The virulence and pathogenicity of bacterial pathogens are related to their adaptability to changing environments. Oneprocess 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. SSRsare found in eukaryotesas 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. In this protocol we validate the computaitonal approaches presented here by sequenciing chemically synthesized oligos and annealed to a form duplexes. These oligos are slightly shorter version of the AAGC SSR found in CP018802.1.