PacBio Long-Read Sequencing and Error Correction

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.