Pacbio rsii instrument

Genomic DNA was extracted from a whole single male P. regina specimen that had been in colony for >15 generations (different colony from above, but same originating location, Indianapolis). DNA library preparation and sequencing was performed according to the manufacturer’s instructions and reflects the P6-C4 sequencing enzyme and chemistry, respectively, at the Icahn School of Medicine at Mount Sinai Genomics Core Facility. 14 % of the input library eluted from the agarose cassette and was available for sequencing. For all cases, this yield was sufficient to proceed to primer annealing and DNA sequencing on the PacBio RSII instrument (Pacific Biosciences, Menlo Park, CA). SMRTcell libraries were placed onto the RSII machine at a sequencing concentration of 150 pM and configured for a 240-min continuous sequencing run. Sequencing was conducted to achieve a 7401 bp subread N50 across a total of 1.5 Gb of data comprised of 268,000 reads on 2 SMRTcells.
Due to the high error rate of reads generated from PacBio sequencing [45 (link)], error correction was performed using the Correct PacBio Reads tool of CLC’s Genome Finishing Module plug-in v1.5.1 (Qiagen Inc) on a local workstation. Additional error correction was also performed using the SMRT Analysis PacBioToCA correction module, with the assistance of the high quality Illumina reads, using default settings.

Top eight samples were taken to perform single-molecule real-time sequencing (SMRT). The cDNAs were synthesized from viral RNAs by reverse transcription using Uni12 and Uni13 primers (Bi et al., 2016 (link)). The PCR was performed using a Phusion High-Fidelity PCR Kit (New England Biolabs) utilizing the barcoded influenza A virus general primers (Supplementary Table S1) (Mei et al., 2016 (link)). The concentration of PCR product was quantified by the Agilent Technologies 2,100 bioanalyzer. The two corresponding volumes of PCR products (containing equal mass of dsDNA) were mixed into one sample and quantified in the bioanalyzer again. About 2–3 μg mixed sample was used to SMRTbell library construction following the 2 kb template preparation protocol (Roberts et al., 2013b (link)). The sequencing was performed on a PacBio RS II instrument (Pacific Biosciences, USA) with one SMRT Cell used for each library, using P6/C4 chemistry with a 4 h movie (Bull et al., 2016 (link)). SMRTbell adapter sequences were removed and circular consensus sequence (CCS) reads were achieved with SMRT Analysis v2.3 (Roberts et al., 2013a ).