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Transposon sequencing (TnSeq) is a next-technology deep sequencing-primarily based totally technique to quantitatively investigate the composition of complicated mutant transposon libraries after stress from selection.
Although this technique may be used for any organism wherein transposon mutagenesis is possible, this bankruptcy describes its use in Mycobacterium tuberculosis. More specifically, the strategies for producing complicated libraries thru transposon mutagenesis, layout of selective stress, extraction of genomic DNA, amplification and quantification of transposon insertions thru next-technology deep sequencing are covered. Determining gene essentiality and statistical evaluation on information accumulated also are discussed.The majority (96%) of the genes we recognized as vital for MAH in vitro had a mutual ortholog withinside the associated and enormously virulent Mycobacterium tuberculosis (Mtb).
However, passaging our library thru a mouse version of contamination discovered a full-size number (54% of overall hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb.
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