Computational protocol: Genome Sequencing and Annotation of Mycobacterium tuberculosis PR08 strain

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Protocol publication

[…] Mycobacterium tuberculosis PR08 (MTB PR08) was isolated from the cerebrospinal fluid (CSF) of a patient diagnosed with both pulmonary and extrapulmonary tuberculosis at a local hospital. The sample was cultured in BBL™ MGIT™ Mycobacterial Growth Indicator Tube supplemented with BBL™ MGIT™ OADC enrichment and BBL™ MGIT™ PANTA™ antibiotic mixture (Becton–Dickinson, Oxford, United Kingdom).Genomic DNA was extracted from MTB PR08 and was sequenced using MiSeq (Illumina, CA, USA), generating a total of 46,013,686 reads in a 300-cycle run. Raw reads were trimmed and assembled de novo using CLCbio (CLC Genomics Workbench version 7.0.3) (CLCbio, Aarhus, Denmark), producing an average coverage of 378 ×. Annotation was performed using the Bacterial Annotation System (BASys) and Rapid Annotation using Subsystem Technology (RAST) online services, and the pathogenicity and virulence genes were determined. The genes were validated using the following external gene annotation databases: TubercuList (http://tuberculist.epfl.ch), UniProtKB (http://www.ebi.ac.uk/uniprot), Virulence Factor Database (VFDB) (http://www.mgc.ac.cn), and TBDatabase (TBDB) (http://www.tbdb.org).The size of the draft genome of MTB PR08 is 4,292,364 bp with a G + C content of 65.2%. It is composed of 214 contigs with 4723 predicted genes of which 4203 were protein coding genes and 48 RNA-encoding genes. A total of 2295 (54.6%) of the protein coding genes were assigned into the Cluster of Orthologous Group (COG) . Using RAST, a total of 393 subsystems were annotated in the MTB PR08 genome ().Comparative analysis of MTB PR08 was performed against two other genomes; PR05 and the reference genome H37Rv. Annotation and comparative genomics analysis of MTB PR08 and the selected reference genomes were carried out using RAST as shown in . In order to identify the functions of the genes that contributed to extrapulmonary TB, the genes were annotated using BASys. Based on the analysis, a putative gene (opcA gene) which may have been involved in extrapulmonary infection was identified. It has been reported to play a role in meningococcal adhesion, invasion of epithelial and endothelial cells and in assembly of Glucose-6-Phosphate-Dehydrogenase (G6PD) , .Comparison of genome sequences using RAST revealed that the closest strains of MTB PR08 are Mycobacterium tuberculosis NCGM2209 (score 521), Mycobacterium tuberculosis UM 1072388579 (score 473) and Mycobacterium tuberculosis NA-A0008 (score 454).This Whole Genome Shotgun project has been deposited at GenBank under the accession number CP010895. […]

Pipeline specifications

Software tools CLC Assembly Cell, CLC Genomics Workbench, BASys, RAST
Application Phylogenetics
Organisms Homo sapiens
Diseases Tuberculosis, Tuberculosis, Pulmonary