Whole genome seq of human: adult male B-Lymphocyte
Library construction have been automated (Perkin-Elmer Janus II) in 96-well plate format. 1ug of genomic DNA is subjected to a series of shotgun library construction steps: (a) Fragmentation through acoustic sonication (Covaris). (b) Enriched fragments are recovered via streptavidin beads and PCR amplified. (c) Since each library is uniquely barcoded, samples can be captured in multiplex. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides is carried out on the HiSeq sequencer. We generate 75 bp paired-end reads. Current throughput is sufficient to complete 6 - 8 multiplexed exomes per lane at high coverage (60 to 80X mean coverage). Our processing pipeline consists of the following elements: (a) Base calls generated in real-time on the HiSeq2500. (b) Demultiplexed, unaligned BAM files produced by Picard ExtractIlluminaBarcodes and IlluminaBasecallsToSam. (c) BAM files aligned to a human reference (hg19hs37d5) using BWA-MEM (Burrows-Wheeler Aligner; v0.7.10)