Whole genome sequencing of urinary tract infection (UTI) microcolonies used for Microcolony-seq
Whole genome sequencing of urinary tract infection (UTI) microcolonies used for Microcolony-seq
Microcolonies were selected from urine of a patient with a UTI infection on LB plate are kept at -80C. Bacteria were regrown in LB at 37C with shaking for 5h. DNA 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 (Concentration of Agarose Gel:1% Voltage:150 V, Electrophoresis Time:40 min). 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.