Sequencing-by-Synthesis: Explaining the Illumina Sequencing Technology

The “sequencing-by-synthesis” technology now used by Illumina was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. They founded the company Solexa in 1998 to commercialize their sequencing method. Illumina went on to purchase Solexa in 2007 and has built upon, and rapidly improved the original technology.

Millions of reactions and the reversible terminators

The Solexa/Illumina sequencing method is similar to Sanger sequencing, but it uses modified dNTPs containing a terminator which blocks further polymerization- so only a single base can be added by a polymerase enzyme to each growing DNA copy strand. The sequencing reaction is conducted simultaneously on a very large number (many millions in fact) of different template molecules spread out on a solid surface. The terminator also contains a fluorescent label, which can be detected by a camera. Only a single fluorescent color is used, so each of the four bases must be added in a separate cycle of DNA synthesis and imaging. Following the addition of the four dNTPs to the templates, the images are recorded and the terminators are removed. This chemistry is called “reversible terminators”. Finally, another four cycles of dNTP additions are initiated. Since single bases are added to all templates in a uniform fashion, the sequencing process produces a set of DNA sequence reads of uniform length.

Although the fluorescent imaging system used in Illumina sequencers is not sensitive enough to detect the signal from a single template molecule, the major innovation of the Illumina method is the amplification of template molecules on a solid surface. The DNA sample is prepared into a “sequencing library” by the fragmentation into pieces each around 200 bases long.  Custom adapters are added to each end and the library is flowed across a solid surface (the “flow cell”) and the template fragments bind to this surface. Following this, a solid phase “bridge amplification” PCR process (cluster generation) creates approximately one million copies of each template in tight physical clusters on the flowcell surface. Illumina has improved its image analysis technology dramatically which allows for higher cluster density on the surface of the flowcell.

Number 1 in the world

Illumina is now the dominant vendor of high-throughput DNA sequencing machines. According to the OmicsMaps website, Illumina has 1145 machines in place worldwide compare to 836 for all other vendors combined! Since Illumina machines generally produce the highest yields, they are responsible for an even larger fraction of the total bases of DNA sequence information assayed worldwide. Illumina currently sells several versions of its DNA sequencing machine, all of which use essentially the same sample prep kits and sequencing technology. The ‘HiSeq’ produces the highest yields (up to 75 billion bases, or Gb) per sample with maximum read length of 100 bases from both ends of each template. A typical ‘HiSeq’ run processes eight samples in 11 days. The ‘MiSeq’ is a less expensive machine that sequences a single sample per run. The ‘MiSeq’ runs more quickly (4-24 hours), but produces much less data per sample- from 200 Mb to 2 GB depending on the number of cycles. The ‘MiSeq’ is capable of reading 150 bp from each end of a template molecule, but 250 bp reads are currently in beta testing.


  1. Ken Gaeddert on October 28, 2017 at 2:18 pm

    I’m looking for some projects for students and wanted to get acquainted with this method, so I had some saliva DNA analyzed for bacterial flora. Universal primers for the 16S rRNA were used and two of the nine variable regions were sequenced for me using the Illumina sequencing by synthesis. In the report from the company which did the sequencing there was a statement that said paired sequencing had been done. Am I correct that this refers to forward and reverse strand sequencing? And was this done for purposes of quality control? (The company was not helpful in answering my questions.)

    Another question that came up refers to a table which listed the number of reads done on each saliva DNA sample. One of the samples is shown below.
    Read Base
    28204 13.37M

    On a followup table, it was described as Tags and Bases:
    Tag Base
    28204 13.37M

    Could you explain that to me?

    Finally, I understand what an OTU is, but why are there different numbers for the different samples? Do those numbers refer to the number of clusters for that OTU?

    sample 1 2 3 4
    OTU1 20 174 106 46

    I know I’m asking alot, so if you don’t feel you want to answer these questions, can you direct me to a resource that would have these answers?

    Thanks for your help!

  2. Alex on June 6, 2016 at 6:48 pm

    How is the fragmentation of DNA accomplished?
    What is the structure of the custom adapters?
    How do the reversible terminator nucleotides work?
    Is each reversible terminator labeled with a different fluorophore? Or are they the same?

    Good article but we want to know more.

    • Leo on December 5, 2016 at 7:33 am

      Agreed. I think the information is withheld intentionally.

Leave a Comment