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Protocol for Whole Genome Digital Expression Profiling using Solexa Technology
Sponsored,vendor-submitted protocol   Published in BioTechniques Protocol Guide 2007 2007 (p.37)

The Solexa Genome Analysis System comprises all hardware, software, procedures and reagents necessary for studying gene expression at the whole genome scale.

a) Preparation of Digital Expression Libraries

Total RNA, converted into double stranded cDNA using standard methods, is cut with a specific restriction enzyme. Sequences adjacent to the cut site are converted into 21 bp tags using a type lls restriction enzyme. Each molecule in the library contains a single tag derived from a single transcript. b) Generating a Clonal Single Molecule Array™ Flow Cell

Flowcell preparation creates clusters of up to 1000 identical copies of each single molecule from the digital expression library. This process involves six steps: Template hybridization, template amplification, linearization, blocking of 3′-ends, denaturation and primer hybridization. All of these steps occur in a fully automated process on a Solexa Station. c) Sequencing-by-Synthesis

The Solexa Genome Analysis System completely automates Sequencing-by-Synthesis (SBS). The flowcell from step b is loaded into the analyzer. Reagents in Solexa's SBS DNA Sequencing kit are placed into the reagent ports and sequencing commences by initiating the instrument software.

Solexa's Sequencing-by-Synthesis utilizes four proprietary nucleotides possessing reversible fluorophore and termination properties. Each sequencing cycle occurs in the presence of all four nucleotides leading to higher accuracy than methods where only one nucleotide is present in the reaction mix at a time.


Figure 1. (Click to enlarge)


d) Image processing and sequence alignment

Solexa's software suite includes the full range of data collection, processing and analysis modules streamlining data analysis with minimal user intervention. Data files are mirrored to an off-instrument computer using a standard software pipeline that sequentially performs image analysis, base-calling, per-base confidence scores and realignment against a reference database. Individual transcript tags are identified and counted (figure 2). Operators can view the data, summary statistics plus access more detailed run metrics and comparisons.


Figure 2. (Click to enlarge)


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