Summary: Illumina's Solexa Sequencing Technology

Here I present a brief overview of Solexa's sequencing-by-synthesis chemistry. The sample prep methods used differ slightly from that used in ABI's SOLiD system, but the basic goals are the same: generate large numbers of unique "polonies" (polymerase generated colonies) that can be simultaneously sequenced. These parallel reactions occur on the surface of a "flow cell" (basically a water-tight microscope slide) which provides a large surface area for many thousands of parallel chemical reactions.

Step 1: Sample Preparation

The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR.

Complete detailed protocols for DNA and small RNA library preparation can be found in the documents provided in the attachments to this post. ("dna_libe_prep.pdf" and "rna_libe_small_prep.pdf", respectively). This process is a fairly straightforward multi-step molecular biology process, however there are many pitfalls that can result in skewed results downstream.

Steps 2-6: Cluster Generation by Bridge Amplification

In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell.

The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface.