Sequence Analysis - Illumina Genome Analyzer

Sequencing-by-Synthesis

Following cluster generation, Genomics Resource staff introduces the flow-cell into the Illumina Genome Analyzer. The Genome Analyzer will perform a series of single-base additions, using a pool of four photocleavable fluorescent nucleotide analogs. Each single-base addition is accompanied by laser excitation of the flow-cell surface, followed by fluorescent signal capture at each of the four emission wavelengths. Each base addition constitutes a "cycle" and Illumina packages their sequencing kits in 18-, 26-, or 36-cycle reactions. Researchers should note that the sequencing error rate increases with cycle numbers. Please contact the Genomics Resource to discuss which kit you should purchase.

Results\Downstream Analysis

A typical 36-cycle sequencing run takes approximately 72 hours to complete. Data is transferred to a user's account on the Research Computing Services server (FRED) in real-time during the course of the run. A full dataset for a 36-cycle run will approach 1 terabyte of data and is not something that can easily be transferred from server to server.

Genomics Resource staff will initiate the standard Illumina analysis pipeline protocols, which convert raw image data to base calls, provide quality scores at each base, and can align sequences to a reference genome. Users are instructed to work with Genomics Resource staff member Ryan Basom at rbasom@fhcrc.org or (206) 667-2747 to arrange for these services. Please note that due to the shear volume of data, image files will be archived to tape following the initial pipeline analysis.

Given the volume of data produced by a single Illumina Genome Analyzer run, as well as the fact that analysis tools are few and not always user-friendly, it is critical for users to partner with or have on staff computationally competent individuals to help with downstream analyses.

The Computational Biology Shared Resource is available for those requiring such individuals. For more information, please contact:

Martin Morgan, PhD

Director, Computational Biology Shared Resource

Phone: (206) 667-2793

Email: mtmorgan@fhcrc.org

Illumina also provides a website containing links to open-source downstream analysis tools.

In all cases, care must be applied in managing and analyzing data files of the magnitude generated when using ultra high-throughput sequencing technologies. Disk space can quickly become full and system performance can suffer. As such, we ask that care be given to such issues when analyzing your data.

For more information, please contact Ryan Basom at rbasom@fhcrc.org or (206) 667-2747.