Pacbio single molecule real time sequencing rsii platform

Two IncHI2 plasmids, namely, pHNLDH400 from a patient with E. coli colonization and pHNYJC8 from chicken meat, were selected for sequencing. Plasmid DNA was purified from the transformants using QIAGEN® Plasmid Midi Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany), and was completely sequenced by PacBio single-molecule real-time sequencing (RSII platform) (Pacific Biosciences, Menlo Park, CA, USA). Raw reads were introduced into the non-hybrid Hierarchical Genome Assembly Process. Analysis and annotation of the resulting sequences were performed using RAC (http://rac.aihi.mq.edu.au/rac/), ISfinder (https://www-is.biotoul.fr//), BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), RAST server (Aziz et al., 2008 (link)), and the Gene Construction Kit 4.0 (Textco BioSoftware, Inc., Raleigh, NC, USA). Plasmids pHNLDF400 and pHNYJC8 were compared with similar oqxAB-bearing IncHI2 plasmids pHXY0908 and pHK0653 obtained from S. Typhimurium, as well as our previously reported plasmids pHNSHP45-2 (Zhi et al., 2016 (link)), and pHNAH67 from E. coli isolate AHC67 (Yang X. et al., 2014 (link)) using BLASTn and BRIG (Alikhan et al., 2011 (link)). The IncHI2 plasmid R478 (GenBank accession number BX664015) served as the reference plasmid for annotation.

Seventeen plasmids were extracted from transformants using a Qiagen Plasmid Midi kit (Qiagen, Hilden, Germany). pHNFP460-1 was sequenced by the use of the Roche 454 GS-FLX platform, and contigs were assembled with 454 GS de novo assembler Newbler version 2.8. pHNZY32 was sequenced using PacBio single-molecule real-time sequencing (RSII platform) (Pacific Biosciences, Menlo Park, CA). Raw sequence data were introduced into the nonhybrid Hierarchical Genome Assembly Process (HGAP version 3). The remaining 15 plasmids were sequenced using Illumina MiSeq technology (Illumina, San Diego, CA). Sequence reads were assembled into contigs by the use of SOAPdenovo version 2.04.
Initial analysis and annotation of contigs were performed using the RAST server (34 (link)), ISfinder (https://www-is.biotoul.fr/), ResFinder (https://cge.cbs.dtu.dk//services/ResFinder/), RAC (http://rac.aihi.mq.edu.au/rac/), BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and Gene Construction kit 4.0 (Textco BioSoftware, Inc., Raleigh, NC). Gaps between contigs were closed by PCR and Sanger sequencing. The replicon types of these plasmids were analyzed using the Plasmid MLST Database (http://pubmlst.org/plasmid/).

The whole genome of rmtB-positive isolates was extracted and sequenced using PacBio single-molecule real-time sequencing (RSII platform) (Pacific Biosciences, Menlo Park, CA). Raw sequence data were introduced into the nonhybrid Hierarchical Genome Assembly Process (HGAP version 4). The plasmid sequences were analyzed and annotated by the RAST server², ResFinder³, PlasmidFinder⁴, and BLAST⁵.