Wednesday, August 22, 2012

Why Next Generation Sequencing?

Next Generation sequencing greatly reduces the amount of space and time needed to sequence by the Sanger method. Next generation sequencing extends the process over millions of parallel reactions instead of being limited to only a few DNA fragments at a time. What used to take months to sequence now only takes a matter of days.

Functional Biosciences uses Illumina sequencing technology for all Next Generation Sequencing projects. Illumina uses proprietary Sequencing by Synthesis technology that supports massively parallel reactions. To do this, copies of single DNA molecules are bound to a solid surface (i.e. flow cell). Fluorescently labeled reverse chain terminators enable detection of single base as they are incorporated into a growing DNA chain. These fluorescent terminators are imaged as the terminator tagged dNTPs are added, and then are cleaved to allow the next base to be incorporated.

Sequencing of genomic material requires random fragmentation performed by sonication or nebulization to lengths necessary to prepare DNA libraries that will be bound to the flow cell. The reads that come off of the sequencer are pieced back together in downstream analysis and are mapped or assembled back to a known genome or transcriptome. There are also times where a reference genome is unavailable and de novo assembly is required.

NGS Applications:

  • Whole Genome Sequencing/Resequencing
  • Small Genome Sequencing/Resequencing
  • smallRNA discovery
  • Metagenomics
  • Gene regulation and protein binding
  • Transcriptomics
  • BAC, Cosmid, Fosmid, etc. Clone checking
  • And much more...