Computational protocol: Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219

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Protocol publication

[…] To exclude the very unlikely possibility that the gerBAC genes had moved to another place in the chromosome, the Δbont Δpyr ΔgerBAC strain and its parent Δbont Δpyr were subjected to whole genome sequence analysis on a Illumina MiSeq sequencer. First, gDNA from both strains was isolated from overnight cultures using the GeneJET Genomic DNA purification kit (Thermo Scientific). DNA purity and concentration was assessed by Nanodrop analysis, gel electrophoresis and Qubit (Thermo Scientific) analysis. Paired-end libraries were constructed using the NEBNext Ultra gDNA library prep protocol with an average insert size of 240 bp, and analyzed on the Agilent BioAnalyzer (VIB nucleomics core, Belgium) resulting in on average 1.2 million reads per sample. Reads were analysed with Qiagen’s CLC Genomics Workbench version 8.5 (http://www.clcbio.com/), and subjected to standard quality control, read trimming and filtering (reads <15 nucleotides were discarded, quality score limit = 0.01, ambiguous nucleotides trim limit = 2), and read mapping to C. botulinum NCTC 11219 reference WGS with accession number JXMR00000000 (using parameters: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.8, similarity fraction = 0.8). This resulted in an average 43-fold genome coverage for the parental strain and 51-fold coverage for the ΔgerBAC mutant. [...] The raw Illumina reads of a previously published diverse set of 152 gIICb strains were retrieved from the NCBI Sequence Read Archive database (Accession number: SRP059342, no corresponding assembly available). The fastq files were processed with BBduk for removal of adapter contamination, trimming (Phred score >28), and size exclusion (read length >50 bp). Each fastq file was subsequently inspected with FastQC for quality control. The genome of each strain was assembled with SPAdes, and the quality of the assembly assessed with QUAST. Functional annotation was done using Prokka with a custom protein database created from strains of the Clostridium genus. The protein content of each genome was finally queried using blastp against the GerX3b subunits present in strain Eklund 17B (gene locus tags for gerC, gerA, gerB: CLL_A3167, CLL_A3168, CLL_A3169). […]

Pipeline specifications

Software tools BBTools, FastQC, SPAdes, QUAST, Prokka, BLASTP
Databases SRA
Application De novo sequencing analysis
Organisms Clostridium botulinum