Illumina Sequencing Platform

We offer a number of different sequencing services at the Icahn Institute and Department of Genetics and Genomic Sciences, including Illumina sequencing platforms.

Sequencing Library Preparation

Each molecule in the DNA library must contain two specific end sequences to be sequenced on the Illumina platform. The DNA strands should also be less than 500 base pairs in length. The first step of library preparation is to fragment large DNA strands—for large genomes this is 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).

Illumina Flow Cell

The Illumina flow cell is the where the sequencing chemistry occurs. The flow cell 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 flow cell. 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. 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 left as molecules immobilized on a 2D surface, that can be sequenced en masse.

The Illumina sequencing method then proceeds as follows:

  • A single base containing a fluorophore and 3' blocking moiety is incorporated by a polymerase.
  • The flow cell is imaged using fluorescent microscopy.
  • The fluorescent and blocking moieties are cleaved, allowing the next base to be incorporated.

This cycle is repeated less than 100 times with minimal loss of signal or accuracy. Since each base contains a different color dye, the nucleic acid sequence is interpreted 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 by the optical resolution of the camera, which allows for extremely high read densities. The HiSeq 2500 instrument contains all the fluidics and optical equipment needed for sequencing, allowing it to accommodate two flow cells and sequence them in parallel.

The flow cells are separated into eight separate lanes. This allows eight different samples to be sequenced. On average, a single lane yields 120-150M reads (TruSeq V3 flow cells). 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.


Read sequences
  per lane

Read length

Instrument run

HiSeq 2500

150 -180 million
reads x 8 lanes

100 bp single read
or paired end

5 days for single read
11 days for paired end


12-15 million
reads x 1 lane

150 bp single read
or paired end

24-36 hours for both

For more information, view our specification sheet.