Both clinical strains had been taken as part of regular care. No ethical approval was necessary for their use. Genome sequencing Bacterial DNA was extracted from the stationary phase cultures grown in LB broth as previously described with slight modification. Briefly, cells had been lysed with SDS followed by sequential treatment method with RNase A and proteinase K. The DNA was to start with precipitated inside a sodium acetate/ethanol resolution, then purified by phenol/chloroform extraction, followed by the ultimate ethanol precipitation. The purified DNA was re suspended in Qiagen Buffer EB for genome sequencing. For Roche 454 pyrosequencing, libraries were ready additional info for whole genome sequencing and eight kb insert paired end sequencing according to the manufac turers protocol.
Samples had been barcoded and sequenced on a FLX Genome Sequencer working with the GS FLX Titanium program. A complete of 353,416 WGS reads/337,391 PE reads and 249,287 WGS/54,954 PE reads had been gener ated for RM13514 and RM13516, respectively. Illumina library planning and sequencing have been run at Ambry Genetics on a HiSeq2000 sequencer. our website A total of 70,096,726 PE reads and 59,857,480 PE reads had been produced for RM13514 and RM13516, respectively. PacBio libraries for constant long read and circular consensus sequence reads have been ready according for the suppliers protocols. PacBio SMRT sequencing was carried out on a PacBio RS instrument applying C2 chemistry. A total of 297,437 CCS reads and 168,165 CLR reads, and 360,848 CCS reads and 134,983 CLR reads were produced for RM13514 and RM13516, respectively.
Genome assembly and gap closure The preliminary assembly was performed as previously described with modifications. Briefly, 454 WGS and PE reads were assembled utilizing Newbler, and contigs broken into 2 kb overlapping fragments. Illumina PE reads have been assembled utilizing VELVET, and contigs broken into 1. five kb overlapping fragments. Polisher software program was then run to evaluate the high quality with the 454 and Illumina assemblies and proofread the consensus sequences. Lastly, GapResolution and dupFinisher packages have been utilised to close gaps and proper mis assemblies to gen erate an preliminary draft assembly, which contained 14 scaf folds composed of 247 contigs, and 12 scaffolds composed of 115 contigs for RM13514 and RM13516, respectively. Optical maps for each strains have been produced making use of the Argus optical mapping method, and the proper contig buy and any mis assemblies were established. We at first closed gaps by primer stroll ing via PCR and Sanger sequencing the amplified region, on the other hand, as a result of the complexity of several repeat regions, this approach was quite tedious and troublesome. We then applied PacBio prolonged reads to shut remaining gaps during the repeat re gions.