Whole genome sequencing of urinary tract infection (UTI) microcolonies used for Microcolony-seq

Urine from a patient with a urinary tract infection was plated on LB agar plate. Microcolonies appeared ~8h after plating. Microcolonies were picked and subjected to Microcolony-seq and to whole-genome sequencing. Genomic DNA of each UTI microcolony was extracted from 1 mL of bacteria using the DNeasy Blood & Tissue Kit (Qiagen). Library preparation and sequencing was carried out by BGI company, China. Concentration of samples was detected by fluorometer or Microplate Reader (Qubit Fluorometer, Invitrogen). Sample integrity and purity were detected by Agarose Gel Electrophoresis. 1μg genomic DNA was randomly fragmented by Covaris. The fragmented genomic DNA were selected by Agencourt AMPure XP-Medium kit to an average size of 200-400bp. Fragments were end repaired and then 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated fragments. PCR products were purified by the Agencourt AMPure XP-Medium kit. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. Samples were deep-sequenced with the DNBseq G400 machine using the 150-cycles paired-end with 350 bp insert size. At least 150X sequencing depth for each nucleotide in each sample was targeted.