Illumina Sequencing Platform
Sequencing Library Preparation
Each molecule in the DNA library must contain two specific sequences at its ends to be sequenced on the Illumina platform. In addition, the DNA strands should be less than 500 base pairs in length. The first step of library preparation is to fragment large DNA strands; for large genomes this done by acoustic disruption.
The required Illumina-specific sequences are known as “adapters” and are introduced by ligating a short olignucleotide sequence to the 5´ and 3´ ends of the DNA sample. PCR is needed to amplify the DNA before sequencing; this is accomplished using primers complementary to the adapters (ligation-mediated PCR).
The Illumina flowcell is the where the sequencing chemistry occurs. It is a glass slide containing small fluidic channels, through which polymerases, dNTPs and buffers can be pumped. The glass inside the channels is decorated with short oligonucleotides complementary to the adapter sequences. The DNA library containing adapters is diluted and hybridized to these oligonucleotides, temporarily immobilizing individual DNA strands onto the flowcell. Library strands are amplified using a “bridge-PCR” strategy employing cycles of primer extension followed by chemical denaturation. Through the in-situ amplification process, the strands are amplified by several thousand-fold. DNA libraries are hybridized to the flow-cell in low molar quantities (6-20pM). This results in a large physical separation between template DNA strands. At the end of amplification, small clusters of identical DNA are molecules immobilized on a 2-d surface that can be sequenced en-mass.
Sequencing by Synthesis
Individual DNA clusters on flow-cell
The Illumina sequencing method proceeds as follows: a single base containing a fluorophore and 3’ blocking moiety is incorporated by a polymerase. Then the flow-cell is imaged using fluorescent microscopy. Afterwards, the fluorescent and blocking moieties are cleaved, allowing the next base to be incorporated. The cycle is repeated >100 times with minimal loss of signal or accuracy. Since each base contains a different color dye; the nucleic acid sequence is inferred by image analysis software as the imaging proceeds. The time for chemistry and imaging of each cycle is approximately an hour. The Illumina platform is limited only by the optical resolution of the camera; this allows for extremely high read densities. The HiSeq 2000 instrument contains all the fluidics and optical equipment needed for sequencing; it can accommodate two flowcells and sequence them in parallel.
Flow-cells are separated into eight separate “lanes”; this allows sequencing of eight different samples. On average, a single lane yields 120-150M reads (TruSeq V3 Flowcells). More than one sample can be sequenced per lane by employing a molecular barcode that is inserted during DNA library creation and read during the sequencing.
The Illumina platform can be used for:
- Transcript profiling and splice variant detection using mRNA-Seq
- Protein-DNA interaction studies using ChIP-Seq
- Whole-exome sequencing using gene capture technology
- Small gene panel sequencing using custom-made gene capture technology
- MicroRNA and small RNA profiling
- Whole-genome sequencing of human DNA
- Genome-wide cytosine methylation scanning using bisulfite converted DNA
HiSeq 2000 Instrument
Illumina MiSeq Instrument
|Read length||Instrument run time|
|HiSeq 2000||120-150 million
reads x 16 lanes
|100 bp single read
or paired end
|5 days for single read
11 days for paired end
reads x 1 lane
|150 bp single read
or paired end
|24-36 hours for both|