biogem

Illumina Sequencing

A recent major paradigm shift in the research community (both at UCSD and nationally) has been the adoption of high throughput next-generation (next-gen) sequencing technologies heralding the digital era of functional genomics and altering the landscape of biomedical investigation. While it is too early to retire microarray-based technologies to the annals of history, the digital information generated by high-throughput sequencing is clearly leading the charge and further development of enabling technologies promises unprecedented insights into gene structure and function.

Illumina 1G Analyzer (Solexa)

The Illumina 1G Analyzer (Solexa) is based on the massively parallel sequencing of millions of fragments using a proprietary clonal single molecule array technology coupled to a novel reversible terminator-based sequencing chemistry. For short sequence reads, the approach has been determined to be highly robust and accurate. Applications in whole-genome association studies, expression analysis, and sequencing in addition to genome wide location studies have been reported. The short individual read lengths, comprising on average 25-75 bp in length, has led to primary applications in re-sequencing where a known reference genome exists rather than sequencing de novo. Paired end reads generate long scaffolds and highly accurate contigs using multiple insert lengths with high library diversity. Using paired-end reads in excess of 2 x 75 bases permits mammalian-scale de novo assembly.

Roche (454 Life Sciences)

The Roche (454 Life Sciences) sequencing technology is capable of sequencing 500 million bases in a ten-hour period, an advance 1000 times more rapid than Sanger's traditional capillary-based electrophoresis method. In this method DNA is amplified using a clonal approach and DNA is sequenced using a micro-fabricated, massively parallel platform. Adaptors are attached to the sheared 800 bp genomic DNA fragments, permitting their capture on tiny beads (28 mm in diameter), and reaction conditions are optimized to favor the attachment of just one fragment per bead. Subsequently oil droplets containing all the requisite reactants for DNA amplification encase the beads, forming an emulsion which maintains each bead distinct from its neighbor. This ensures uncontaminated amplification of approximately 10 million copies of the initial fragment. The beads are then dispensed into the open wells of a fiber optic slide and 'pyrosequenced' (which detects extension via luciferase based real time monitoring of pyrophosphate release). This generates sequencing reads 500 bp in length making it an effective tool for de novo sequencing.

Illumina's sequencing technology is now available at BIOGEM. Roche 454 sequencing technology is now available at the VMRF GeneChip Microarray Core

Illumina GA Applications

The Genome Analyzer (GA) system uses massively parallel sequencing of billion of fragments using a proprietary reversible terminator-based sequencing technology that can perform a number of applications, including DNA-sequencing, digital gene expression, small RNA sequencing, chIP-sequencing and bisulfite sequencing.

More information on the Genome Analyzer platform can be found at www.illumina.com/sequencing/

Last Updated March 2009
By Dr. Gary Hardiman