Computational protocol: Complete Genome Sequencing of Mycobacterium bovis SP38 and Comparative Genomics of Mycobacterium bovis and M. tuberculosis Strains

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Protocol publication

[…] Extracted DNA was sent to the Genomics Core Facility of Purdue University, USA for sequencing. Briefly, the quality and concentration of the DNA were measure by Nanodrop 2000C (Thermo Fischer Scientific, Massachusetts, USA) and 0.8% agarose gel using a mass ladder. DNA was sheared into 800 bp (base pairs) fragments using the Hydroshear DNA Shearing Device (Genomic Solutions, Michigan, USA) to create fragments for paired end libraries. Sheared DNA was analyzed using a DNA High Sensitivity Chip in an Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA), resulting in average fragment size of 804 bp. A paired-end genomic library was then constructed using TruSeq DNA PCR-free sample preparation kit (Illumina, California, USA) and 20% of a lane (Illumina v3 chemistry) of Illumina HiSeq2500 was used to sequence it.De novo assembly of the sequenced reads was performed using ABySS software (Simpson et al., ) and CLC Genomics Workbench 8 (QIAGEN, Venlo, Holland). Additionally, reads were mapped to a reference genome (M. bovis strain AF2122/97—NC_002945.3) with CLC Genomics Workbench 8 (QIAGEN). ABySS generated contigs were ordered with Projector 2 (van Hijum et al., ) using the same reference genome. Contigs showing clear overlaps based on Smith-Waterman alignment, without repetitive sequences, were manually joined. Remaining gaps were closed using complementary information from CLC Genomic Workbench's de novo and reference mapping assemblies and/or using PCR followed by Sanger sequencing. Gene prediction and annotation was performed by NCBI Prokaryotic Genome Annotation Pipeline (PGAP). […]

Pipeline specifications

Software tools ABySS, CLC Genomics Workbench, Projector 2, PGAP
Application De novo sequencing analysis
Organisms Mycobacterium bovis, Bos taurus, Homo sapiens, Mycoplasma bovis, Bacteria, Corynebacteriales
Diseases Tuberculosis