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Next Generation Sequencing Core

Technology

New technologies for DNA sequencing referred to as “next generation” or “deep” sequencing are currently being applied to many genomics applications. Our facility at Scripps Research operates an Illumina HiSeq system and a Roche 454 GS junior system.

The Illumina HiSeq system utilizes a “sequencing by synthesis” method. Each flow cell is divided into eight separate lanes allowing for up to 8 separate samples to be run simultaneously. Additional levels of multiplexing can be accomplished using barcoded adapter sequences. Read lengths of 50 to 300 bases are generated by hybridizing a library with ligated adapter sequences to the glass surface within the flow cell. The flow cell surface has covalently attached oligonucleotide probes that are complementary to the adapters on the library. Next, a polymerase amplification step is used to amplify the individual hybridized library fragments in spatially distributed “clusters” on the flow cell surface. Fluorescently labeled nucleotides with blocked 3’-OH are used in a series of polymerase-mediated single base extension steps with the 3’ hydroxyl groups de-blocked and fluorescent labels cleaved off after each round of extension. By collecting fluorescent images of the flow cell after each extension reaction, the incorporated nucleotide at each cluster can be identified. After the sequencing run, cluster sequences are determined and quality filtering is applied.

The Roche 454 GS junior system is a pyrosequencing-based method. Libraries are amplified by emulsion PCR on the surface of individual agarose beads. Each bead amplifies a single unique DNA fragment. Beads with amplified fragments are then distributed onto the surface of the 454 picotiter plate. Pyrosequencing is then performed and light flashes from luciferase activity are analyzed to determine which nucleotides are sequentially incorporated. The Roche system can generate longer reads (300-500 bases) and with a typical output of 20-100K reads per run.