Figure 1. Mapping and quantifying tens of thousands of transposon insertion strains by high-throughput insertion sequencing (INSeq).

(A) A negative selection scheme for identification of genes required for colonization in vivo. Mutants in genes important for competitive growth (red) are expected to decrease in relative abundance in the output population. (B) Preparation of an INSeq library. Genomic DNA is extracted from the mutagenized bacterial population, digested with MmeI, and separated by polyacrylamide gel electrophoresis (PAGE). Transposon-sized fragments are appended with double-stranded oligonucleotide adapters by ligation. Limited cycles of PCR create the final library molecules for sequencing. (C) Map of insertion sites in the B. thetaiotaomicron genome. An arrow marks the origin of replication. (D) Reproducibility of library preparation and sequencing protocols. Technical replicates were prepared and sequenced from a single transposon mutant population. Each point represents the abundance of insertions in a single gene; the coefficient of determination, R², on log-transformed abundance values is 0.92.