Computational protocol: Sequence analysis for detection of drug resistance in Mycobacterium tuberculosis complex isolates from the Central Region of Cameroon

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Protocol publication

[…] Purified PCR products were sequenced with the same primers using the ABI’s Big dye terminator kit (Applied Biosystems, USA) according to the manufacturer’s instructions. At each locus, both forward and reverse primers at each locus were included in order to maximize the coverage of the amplified gene fragment, and the reproducibility of the results. Sequencing reactions include 1 μl big dye, 2 μl sequencing buffer, 0.5 μl of each 2.5 μM primer, a volume of PCR template corresponding approximately to 2–3 ng of DNA, and sufficient distilled water for obtaining a 10 μl final volume. Unincorporated terminators were removed by treatment on a sephadex column. The obtained sequences were aligned using the assembling application of vector NTI (Invitrogen) and CodonCode Aligner, and polymorphisms detection was achieved by comparison with the published M. tuberculosis H37Rv sequence. […]

Pipeline specifications

Software tools Vector NTI Advance, CodonCode Aligner
Application Protein sequence analysis
Organisms Mycobacterium tuberculosis, Homo sapiens
Diseases Tuberculosis, Tuberculosis, Pulmonary
Chemicals Ethambutol, Rifampin