Pacbio rs 2 sequencing technology

DNA sequencing template was obtained from sheared genomic DNA using the Pacific Bioscience 10 kb SMRTbell library template preparation kit per the manufacturer's instructions (Pacific Biosciences, Menlo Park, CA, USA). The quality sizing analysis of DNA library was validated by Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Inc., Santa Clara, CA, USA) prior to sequencing. PacBio RS II sequencing technology (Pacific Biosciences) was used as the sequencing platform. P4 chemistry was utilized, and the prepared library was sequenced on four single-molecule real-time (SMRT) cells.

(i) Illumina MiSeq sequencing. Total DNA was extracted using a Qiagen genomic tip-100 (anion-exchange tip) (Qiagen, Valencia, CA) according to the kit manual, with minor modifications. Briefly, 6 ml of an overnight culture with vancomycin at 25 μg/ml, fusidic acid at 25 μg/ml, and rifampin at 50 μg/ml was used for each DNA sample. To break up the cells, 80 μl of 100 mg/ml lysozyme was used with 2 h of incubation at 37°C. The DNA samples were diluted to 0.3 ng/μl, and 5 μl was used for library generation using a Nextera XT DNA sample preparation kit and Nextera XT index primers (Illumina, San Diego, CA). Sufficient sample was diluted to 600 μl to provide a 15- to 20-pmol multiplexed library and sequenced on an Illumina MiSeq V2 instrument as 2×150 bp paired-end reads.
(ii) PacBio single-molecule sequencing. DNA was isolated as described above for Illumina sequencing, and 10 μg of high-quality DNA was used to make large insert libraries (10 kb) to be sequenced using the Pacific Biosciences (PacBio) RS II sequencing technology (Pacific Biosciences, Menlo Park, CA). For each sample, we used one PacBio RS II SMRT cell.