Extended Data Fig. 8: Development and analysis of Tn-seq.

a, Schematic of the V. cholerae transposon end sequences. The 8-bp terminal sequence of the transposon is boxed and highlighted in light yellow. Mutations generated to introduce MmeI recognition sites are shown in red letters, and the resulting recognition site is highlighted in red. Cleavage by MmeI occurs 17–19 bp away from the transposon end, generating a 2-bp overhang. b, Comparison of integration efficiencies for the wild-type and MmeI-containing transposon donors, determined by qPCR. Labels on the x axis denote which plasmid was transformed last; we reproducibly observed higher integration efficiencies when pQCascade was transformed last (crRNA-4) than when pDonor was transformed last. The transposon containing an MmeI site in the transposon ‘right’ end (R∗-L pDonor) was used for all Tn-seq experiments. Data are mean ± s.d. for n = 3 biologically independent samples. c, Plasmid expression system for Himar1C9 and the mariner transposon. d, Scatter plot showing correlation between two biological replicates of Tn-seq experiments with the mariner transposon. Reads were binned by E. coli gene annotations, and a linear regression fit and Pearson linear correlation coefficient (r) are shown. e, Schematic of 100-bp binning approach used for Tn-seq analysis of transposition experiments with the V. cholerae transposon, in which bin 1 is defined as the first 100 bp immediately downstream (PAM-distal) of the Cascade target site. f, Scatter plots showing correlation between biological replicates of Tn-seq experiments with the V. cholerae transposon programmed with crRNA-4. All highly sampled reads fall within bin 1, but we also observed low-level but reproducible, long-range integration into 100-bp bins just upstream and downstream of the primary integration site (bins −1, 2 and 3). g, Scatter plot showing correlation between biological replicates of Tn-seq experiments with the V. cholerae transposon programmed with a non-targeting crRNA (crRNA-NT). h, Scatter plot showing correlation between biological replicates of Tn-seq experiments with the V. cholerae transposon expressing TnsA-TnsB-TnsC-TniQ but not Cascade. For f–h, bins are only plotted when they contain at least one read in either dataset.