Illumina: NextSeq 500

Reversible Dye Termination Sequencing by Synthesis Technology


Launched in January 2014, the NextSeq 500 is the newest platform to be added to Illumina’s NextGen Sequencing portfolio (in addition to the HiSeq X10). The NextSeq 500 offers middle and high throughput options in the convenience of a desktop sequencing package. Like other Illumina sequencing platforms, the NextSeq 500 also uses reversible dye termination sequencing by synthesis technology. However, the NextSeq 500 uses a 2-channel (i.e. 2 dye) sequencing technology as opposed to its predecessors that use 4-channel. This advancement in sequencing technology allows for a significant reduction in cycle and data processing times.

The basic principle behind how Illumina sequencing works is fluorophore detection. First, libraries undergo bridge amplification to generate clonal clusters of library molecules directly on the surface of the sequencing flow cell. The nucleotides used during NextSeq 500 sequencing are uniquely labeled as compared to 4-channel sequencing instruments (like the MiSeq or HiSeq 2500): “C” nucleotides are labeled with a red fluorophore, “T” nucleotides are labeled with a green fluorophore, “A” nucleotides are labeled with both a red and green fluorophore (shown as “yellow”) and “G” nucleotides are not labeled at all. However, all 4 nucleotides are still chain terminating with their terminating properties reversible.

During sequencing, all nucleotides are pushed through the flow cell lanes and allowed to anneal to the library clusters, with only one nucleotide annealing at a time. After annealing, the excess nucleotides are washed away and the fluorophore emissions from each cluster are imaged. In the case of the NextSeq 500, only red and green images are taken. An enzymatic reaction will cleave off the fluorophore and the next round of nucleotide annealing will take place. Each nucleotide is coded by a different “color” emission (or non-emission in the case of “G” nucleotides) and the resulting data is converted by the software into nucleotide sequence.

NextSeq 500 Platform Specifications

The sequencing specifications of the NextSeq 500 are shown in the table below. This table was taken directly from the Illumina Sequencing Specification sheet. The full document can be accessed here.

Illumina provides a Sequencing Coverage Calculator for the NextSeq 500.  Download the file below (along with Illumina’s Technical Application Note) to help you with your experimental design.


Become a member of myIllumina to get access to assay design tools, product updates and more!

Illumina Sequencing Applications

There are several supported applications for Illumina DNA, RNA and epigenetics sequencing. Click the links below to get more information on their most popular applications.