Protocol for Whole Genome Sequencing using Solexa Technology

The Solexa sequencing platform comprises all hardware, software, procedures and kitted reagents for DNA sequencing at the whole genome or repetitive regional scale.

a) Preparation of full diversity libraries of whole genomes

Whole genomic DNA is randomly fragmented and end-repaired. Solexa's oligonucleotide adapters are ligated onto the fragments, yielding a fully-representative genomic library of DNA templates without cloning.

b) Generating a Clonal Single Molecule Array™ Flow Cell

Single molecule clonal amplification involves six steps: Template hybridization, template amplification, linearization, blocking 3' ends, denaturation and primer hybridization.

Flowcell preparation is fully automated on a Solexa Cluster Station creating up to 1000 identical copies of each single molecule achieving densities of up to 10 million clonal clusters per square centimeter.

c) Sequencing-by-Synthesis

The Solexa 1G Genetic Analyzer completely automates Sequencing-by-Synthesis (SBS). The flowcell from step b is loaded into the analyzer. Reagents and buffers in Solexa's SBS 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.

d) Image processing and sequence alignment

Developed in collaboration with leading researchers, Solexa's software suite includes the full range of data collection, processing and analysis modules to streamline collection and analysis of data with minimal user intervention. After sequencing has completed, the data files are mirrored to an off-instrument computer for analysis using a standard pipeline of software that sequentially performs image analysis, generation of base-calls, per-base confidence scores and realignment against a reference database. The operator can view the data, summary statistics and error analysis plus access more detailed run metrics and comparisons.