Pacbio rsii smrt platform

We assembled the beaver genome using a strategy whereby a primary assembly was generated using moderate-coverage, uncorrected long reads from the PacBio RSII SMRT sequencer. Long read sequencing using the PacBio RSII SMRT platform was performed at the Ontario Institute for Cancer Research (OICR) in Toronto. PacBio SMRTbell libraries were constructed from purified genomic DNA from fresh beaver blood leukocytes. The DNA shearing step was omitted since pulsed-field gel analysis showed that genomic DNA isolated from Ward already had a broad size distribution with a peak mode length of ∼40 kb, similar to the recommended size for library construction. Libraries were prepared from 15 μg of genomic DNA using the SMRTbell Template Prep Kit 1.0, according to the manufacturer’s instructions for 20 kb insert templates. SMRTbell libraries were size-selected using the BluePippin System (Sage Science, Beverly, MA) with a lower cut-off of 15 kb. We performed whole-genome SMRT sequencing using 80 flow cells, running the current P6/C4 chemistry with a 6 hr movie-time, and used PacBio’s P-Filter Module protocol to process raw data to remove library adaptors and redundant or low-quality sequence reads. Each of the 80 flow cells produced an average of 1 Gb of filtered PacBio reads with average read length of ∼13–14 kb, and together represented ∼30-fold coverage of the beaver genome.