Real time sequencer rtii

HapMap normal human sample NA12878 is a very well-studied sample used for assessing both germline DNA and RNA analysis. Since high-confidence genomic variants have been made available for this sample, it offered a great opportunity for assessing RNA-seq variant-calling. PacBio and Illumina sequences were obtained from an earlier study^{10 (link)}. It can be found in the NCBI Sequence Read Archive (accession no. SRP036136). Further data are available at http://stanford.edu/~htilgner/2014_PNAS_paper/utahTrio.index.html. The data set includes 115.4 million 101-bp paired-end reads from an Illumina Hi-Seq 2000 sequencer and 715,902 CCS long reads from a Pacific Biosciences real-time sequencer RTII. The mean length of the long reads was 1,188 bp with a maximum of 6 kbp.

SMRTbell libraries were constructed by using the uniform mixed RNA form roots, stems, leaves and flowers and Pacific Biosciences’ 1.0 template prep kit (part 100-259-100) according to the manufacturer’s instructions. The synthesized cDNA was run on an agarose gel and 2 separate size ranges were fractionated: 1–2 kb, 2–3 kb. Each size fraction was extracted from the gel and treated according to Pacific Biosciences’ template preparation and sequencing protocol. The DNA/polymerase binding kit P5 (part 100-256-000) and v2 primers were used to make SMRTbell templates bound to polymerases. The polymerase–template complexes were bound to magbeads using Pacific Biosciences’ Magbead binding kit (part 100-133-600), and SMRT sequencing was then carried out on Pacific Biosciences’ real-time sequencer RTII by using DNA Sequencing Reagent 3.0 (part 100-254-800). All movie lengths were set to 240 min for each SMRT cell.