Transposon insertion sequencing

Transposon insertion sequencing (Tn-seq) was performed as described previously [65–67]. Libraries of >100,000 independent transposants were separately generated in wild-type and a ΔlytE mutant. Genomic DNA was extracted from each and digested with MmeI, followed by adapter ligation. Transposon-chromosome junctions were amplified by PCR (17 amplification cycles). PCR products were pooled, gel-purified, and sequenced on the Illumina HiSeq platform using TruSeq reagents (Tufts University TUCF Genomics facility). Reads were mapped to the B. subtilis 168 genome (NCBI {"type":"entrez-nucleotide","attrs":{"text":"NC_000964.3","term_id":"255767013","term_text":"NC_000964.3"}}NC_000964.3), tallied at each TA site, and genes in which reads were statistically underrepresented were identified using the Mann Whitney U test and by visual inspection using Sanger Artemis Genome Browser and Annotation tool [68].