Transposon insertion mapping with PIMMS – Pragmatic Insertional Mutation Mapping System

Abstract

The PIMMS (Pragmatic Insertional Mutation Mapping System) pipeline has been developed for simple conditionally essential genome discovery experiments in bacteria. Capable of using raw sequence data files alongside a FASTA sequence of the reference genome and GFF file, PIMMS will generate a tabulated output of each coding sequence with corresponding mapped insertions accompanied with normalized results enabling streamlined analysis. This allows for a quick assay of the genome to identify conditionally essential genes on a standard desktop computer prioritizing results for further investigation.Availability: The PIMMS script, manual and accompanying test data is freely available at https://github.com/ADAC-UoN/PIMMS

PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus

“…Sequence data was analyzed and mapped back to the original genome using the PIMMS bioinformatic pipeline (Blanchard et al, 2015). Comparative analysis indicated that the number of sequence reads that included 23 bp from either terminus of the insertion sequence (Matched Reads; Table 1 ) was similar for the EcoR1 and HindIII digested samples (62.3% and 62.9%, respectively).…”

“…A bioinformatic pipeline (Blanchard et al, 2015) was used to map the sequence directly adjacent to the IS S1 terminus to the S. uberis reference genome (accession number AM946015; Ward et al, 2001). This revealed that between 39 and 25% of the matched reads mapped unambiguously to the source genome.…”

“…The PIMMS pipeline (Blanchard et al, 2015) was used to process the reads and map them to the S. uberis 0140J reference genome [accession number AM946015 (Ward et al, 2009)]. Briefly, each sequence read was assessed for the presence of the terminal portion of ISS1 [accession number (of pGh9:ISS1) EU223008.1] and once identified, the remaining sequence was analyzed for quality.…”

“…These each require a series of complex steps to produce mutants and isolate DNA fragments flanking insertions and in some cases specialized sequencing procedures are required to generate the final data set. Various bioinformatic approaches and predictive modeling strategies have been used to analyze the vast amounts of data produced from such protocols (Zomer et al, 2012; Chao et al, 2013; Pritchard et al, 2014; Blanchard et al, 2015). …”

“…The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al, 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria.…”