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Wednesday, August 2 • 15:50 - 16:10
"Mutation grade" bacterial sequences: the Bacillus cereus group as an example

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Analysis of point mutations in bacteria using NGS technologies needs very precise sequence of wild type (WT) strains to be used as templates for the variant analysis. Our experience in analysis of mutations in several strains of the Bacillus cereus group (B. cereus sensu stricto, B. thuringiensis, B. weihenstephanensis) indicated that the available Genebank sequences, produced with different technologies, are generally unsuitable for this purpose. Thorough scrutiny of the WT strain sequence must be done in parallel. As an example the available sequence of the B. cereus ATCC14579T, produced by Sanger technology, contains about 2000 sequencing errors. Read alignment with more recent sequencing data, like that of B. thuringiensis 407, produced using 4-5-4 technology, produces about 200 variations that completely hampers direct mutation analysis. The most suitable sequence that we analyzed was that of B. weihenstephanensis KBAB4 strain containing to our estimation about 20 errors. The available sequence of this strain, mainly assembled after Sanger sequencing, was refined before submission with Illumina technology. We therefore propose a concept of so-called "mutation grade" sequence that is the one containing less than 1-2 errors per Mb (0.0001%) in the whole resolvable area and thus directly suitable for point mutation mapping. Our experience in generating such sequences and point mutation analysis for the B. cereus ATCC14579T and a conjugation competent B. weihenstephanensis strains will be presented in this talk. It must be noted that generation of absolutely error-free sequence is impossible in practice, even for bacteria. This is mostly due to "unresolvable" locations in the genomes, usually containing complex repetitive sequences. Such locations are specific for each bacterial group and should be taken into account during variation analysis. The mutations in B. cereus ATCC14579 were mapped in the contexts of selection of psychrotolerant clones and conjugation experiments. Our study demonstrates that the level of sequence variation existing between strains, used in different laboratories or produced in "blind" experiments, like conjugation, is about 2-4 mutations/genome. Although looks negligible, such variation level can be frustrating and leading to misinterpretation of data when analyzing the bacterial sequences with so high precision. The sequence of conjugation competent B. weihenstephanensis strain was completed using Illumina technology, different assemblers, including spades that produced the minimal number of contigs, and subsequent semi-manual sequence verification assisted with complete KBAB4 sequence for gap closure. Mapping of "blind" mutations accumulated during conjugation experiments showed that the produced sequence of this strain is of "mutation grade" quality. The work was partially supported by the National French Research Agency (ANR project PathoBactEvol).


Wednesday August 2, 2017 15:50 - 16:10
Graduate School of Management Building, room 309 Volkhovskiy Pereulok, 3, St. Petersburg, Russia