DNA Analysis Facility

Sample Pipeline

Samples to be sequenced on the GA go through several stages. At present we ask the investigator to perform library prep.

Library Prep

There are several sample preparation methods, which allow the GA to be used for a variety of applications. For genomic sequencing and resequencing, genomic DNA is sheared into random fragments and capped with adapter sequences. For digital gene expression studies, RNA is isolated, adapters are ligated, and RT-PCR is performed to generate template for cluster generation and eventual sequencing. Please see our protocols section for more information.

Cluster Generation

Once a sample library is created, the Cluster Station hybridizes samples to a flow cell and amplifies them for sequencing. The process is as follows:

  1. DNA is hybridized to the flow cell.
  2. Template is amplified making dsDNA clusters.
  3. dsDNA clusters are linearized.
  4. The 3 prime ends of the linearized clusters are blocked to prevent nonspecific sites from being sequenced.
  5. Clusters are denatured
  6. Sequencing primers are hybridized onto the linearized blocked clusters.

Sequencing

The Genome Analyzer sequences clustered template DNA with reversible terminators and removable flourescence using a sequencing-by-synthesis technology. This provides high accuracy through homopolymer regions. Massively parrallel sequencing of >36 bp reads of over 50 million clusters results in 1.5 Gb of single-read data in 2.5 days and 3 Gb of paired-end sequence data in 5 days with high accuracy at greater than >3× genome coverage.