Figure 1E.3.2.

Flowchart depicting Tn-seq, from library construction to massively parallel sequencing of transposon-chromosome junctions. (A) A gene-disruption library is constructed by transposing the mini-transposon magellan6 into bacterial genomic DNA in vitro and subsequently transforming a bacterial population with the transposed DNA. The result is a pool of strains where each bacterium contains a single transposon randomly inserted in its genome. DNA is isolated from a portion of the bacterial pool (t₁); another portion is used to seed a culture on which selection is performed (in vitro or in vivo); after selection, bacteria are recovered and DNA is isolated again (t₂). (B) To accomplish sequencing of only the regions flanking magellan6 insertions, DNA from both time points is digested with MmeI, which binds a sequence in the magellan6 terminal inverted repeats but cuts 20 bp downstream, leaving a two-base overhang to which an adapter is ligated. A PCR is performed with one primer having complementarity to the adapter and the other complementary to the inverted repeat sequence. (C) The resulting PCR product is 120 bp long, with approximately 20 bp of bacterial specific DNA flanked by Illumina specific sequences needed for sequencing. After sequencing, different samples are split based on barcode sequence, and the bacteria-specific reads are mapped to the genome and counted for each insertion, thus allowing fitness to be calculated.