Biochemistry and Pharmacology - Research Publications
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ItemISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data(BIOMED CENTRAL LTD, 2015-09-03)BACKGROUND: Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data. RESULTS: ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots. CONCLUSIONS: ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.
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ItemEvidence of microevolution of Salmonella Typhimurium during a series of egg-associated outbreaks linked to a single chicken farm(BMC, 2013-11-19)BACKGROUND: The bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most frequent causes of foodborne outbreaks of gastroenteritis. Between 2005-2008 a series of S. Typhimurium outbreaks occurred in Tasmania, Australia, that were all traced to eggs originating from a single chicken farm. We sequenced the genomes of 12 isolates linked to these outbreaks, in order to investigate the microevolution of a pathogenic S. Typhimurium clone in a natural, spatiotemporally restricted population. RESULTS: The isolates, which shared a phage type similar to DT135 known locally as 135@ or 135a, formed a clade within the S. Typhimurium population with close similarity to the reference genome SL1334 (160 single nucleotide polymorphisms, or SNPs). Ten of the isolates belonged to a single clone (<23 SNPs between isolate pairs) which likely represents the population of S. Typhimurium circulating at the chicken farm; the other two were from sporadic cases and were genetically distinct from this clone. Divergence dating indicated that all 12 isolates diverged from a common ancestor in the mid 1990 s, and the clone began to diversify in 2003-2004. This clone spilled out into the human population several times between 2005-2008, during which time it continued to accumulate SNPs at a constant rate of 3-5 SNPs per year or 1x10-6 substitutions site-1 year-1, faster than the longer-term (~50 year) rates estimated previously for S. Typhimurium. Our data suggest that roughly half of non-synonymous substitutions are rapidly removed from the S. Typhimurium population, after which purifying selection is no longer important and the remaining substitutions become fixed in the population. The S. Typhimurium 135@ isolates were nearly identical to SL1344 in terms of gene content and virulence plasmids. Their phage contents were close to SL1344, except that they carried a different variant of Gifsy-1, lacked the P2 remnant found in SL1344 and carried a novel P2 phage, P2-Hawk, in place SL1344's P2 phage SopEϕ. DT135 lacks P2 prophage. Two additional plasmids were identified in the S. Typhimurium 135@ isolates, pSTM2 and pSTM7. Both plasmids were IncI1, but phylogenetic analysis of the plasmids and their bacterial hosts shows these plasmids are genetically distinct and result from independent plasmid acquisition events. CONCLUSIONS: This study provides a high-resolution insight into short-term microevolution of the important human pathogen S. Typhimurium. It indicates that purifying selection occurs rapidly in this population (≤ 6 years) and then declines, and provides an estimate for the short-term substitution rate. The latter is likely to be more relevant for foodborne outbreak investigation than previous estimates based on longer time scales.