Automated High-Throughput DNA Sequencing

Genomic DNA was extracted from whole blood or buffy coats using Qiagen kits (Qiagen, Chatsworth, CA, USA) or salting out procedures described previously [20 (link),21 (link)]. 1 ug of genomic DNA was sent to the Core lab at Northwest Genomics Center at the University of Washington for sequencing. The quality and integrity of DNA was checked at the Core lab using Agilent’s Analyzer and Tape Station reagents before target capture and library preparation. Library construction and custom capture have been automated (Perkin-Elmer Janus II) in a 96-well plate format. The purified DNA was subjected to a series of shotgun library construction steps, including fragmentation through acoustic sonication (Covaris), end-polishing and A-tailing, ligation of sequencing adaptors, and PCR amplification with 8 bp barcodes for multiplexing. Libraries undergo capture using the Roche/Nimblegen SeqCap EZ custom designed probe. Prior to sequencing, the library concentration was determined by triplicate qPCR and molecular weight distributions verified on the Agilent Bioanalyzer. Barcoded libraries were pooled using liquid handling robotics prior to clustering (Illumina cBot) and loading. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on the HiSeq sequencer.