The initial step in the sequencing process is the preparation of the library. A library is a representation of the source nucleic acid (RNA or DNA) in a collection of short fragments generated randomly by chemical degradation or mechanical shearing.The preparation of the library is a multi-step process.
For genomic DNA, the process of preparing the library can be visualized by the following.
The unique feature of sequencing with the Illumina HiSeq 2000 is that the amplification of individual templates is automated and performed on a flowcell. A flow cell resembles a microscope slide with 8 channels running lengthwise.
When the process is complete, each flowcell will have as many as 180 million individual readable clusters in one lane on the flowcell. Each flowcell has 8 lanes, so the total reads for a single run will exceed 1.4 billion.
Sequencing is performed by proprietary chemistry using reversible dye terminators. Each sequencing pass incorporates one of the four nucleotides into the growing chain. At the end of the pass, five images are captured - one for focusing and one each for the four nucleotides, which are distinguished by their fluorescence characteristics. Software calculates the location of each cluster and makes a base call for the cluster. Sequencing runs are from 50 to 100 bases and run from 3 to 12 days. All libraries are constructed with barcodes, so multiple samples can be run in each lane.