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Pipeline publication

[…] erol and 0.025% (v/v) Tween 80. The handling of strains was done in BSL-3 facilities at either Statens Serum Institut, Denmark or the Center for Infectious Disease Research, WA, USA., Six weeks after the third immunization animals were infected with 100 viable bacilli by the aerosol route, with M.tb Erdman, M.tb DK9417, M.tb DK9897 or M.tb DK9897 complemented with pKG18 (DK9897::pKG18). Six weeks after infection, mice were sacrificed and organs homogenized in PBS for bacterial enumeration, as described in, and flow cytometry analysis., MIRU-VNTR 24-locus genotyping and DNA extraction from bacterial cultures were performed as previously described. A minimum spanning tree was created with the MIRU-VNTRplus database website. MIRU-VNTR clusters were defined as strains with identical genotyping patterns and clonal complexes by a maximum difference of two loci. Genomic DNA of M.tb DK9897 was sequenced, mapped and variance analyzed by Eurofins, Ebersberg, Germany. Sequencing was done using the MiSeq system (Illumina, San Diego, California, USA) with 300 base pair (bp) paired-end reads from a Nextera DNA library, yielding a total of 2359 Mbp calls. Low-quality bases (Phred quality score <20) and clipped reads shorter than 40 bp were removed using Trimmomatic (v0.32) using the sliding window approach (size 4 bp) resulting in a mean read length of 225 bp. The trimmed reads were mapped to the M.tb strain H37Rv genome (GenBank ID: NC_000962.3) using the Burrows–Wheeler Aligner BACKTRACK (v0.6.2-r126) resulting in a mean genome coverage of 365. Variant analysis was performed using VarScan (v2.3) aligning reads with a quality of at least 1 (Phred quality score). The variants were classified into categories: LOW (synonymous variant), MODERATE (missense variant, in frame insertion/deletion) and HIGH (stop lost/gained, frameshift insertion/deletion). Results were analyzed using Excel (v15.0.4885.1000). The complete annotation of the M.tb DK9897 genome is located under GenBank accession no. CP018778., The presence and integrity of twelve ESX-1 related genes were analyzed in a panel of M.tb complex strains and clinical isolates for which the genomes had been completely sequenced. These organisms were selected from the Nucleotide collection database of the National Center fo […]

Pipeline specifications

Software tools MIRU-VNTRplus, Trimmomatic, VarScan