Illumina Sequencing by Synthesis

Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process, through to data analysis on the BaseSpace® Sequence Hub.

Want more details? Download an introduction to Illumina next-generation sequencing technology for an in-depth look at SBS chemistry.

See how clinical whole-genome sequencing is giving hope to families and providers seeking answers for children with rare and undiagnosed genetic diseases:

For more information on the applications and advantages of SBS technology, visit

Drill down further with this Technology Spotlight on Illumina Sequencing Technology

Review historic milestones in the development of Illumina next-generation sequencing genetic sequencing technologies.

A global genomics leader, Illumina provides comprehensive next-generation sequencing solutions to the research, clinical, and applied markets. Illumina technology is responsible for generating more than 90% of the world’s sequencing data.* Through collaborative innovation, Illumina is fueling groundbreaking advancements in oncology, reproductive health, genetic disease, microbiology, agriculture, forensic science, and beyond.
*Data calculations on file. Illumina, Inc., 2015.

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Comment (235)

  1. what does it mean 'the number of cycles determines the length of the read?' at 2:45 .. I thought all fragments are read at the same time.. Please someone help 😣​

  2. I have a question. It seems during the first read all the two types of oligos have been occupied through bridging amplification. Why there are free oligos to be used for bridging during the second read?

  3. Hello, thank you for the nice video. It is indeed very helpful.
    One question that has been addressed before by another person (@quickfastgoninja) in the comments:
    Considering that both oligo complements ('purple' and 'blue') are present in the flow cell:
    what is preventing the "blue" sequences from hybridizing when the DNA is flowed across the cell? From the animation, it seems that purple:purple hybridization would be equally as likely as simultaneous blue:blue hybridization. Are the "blue" adapters somehow blocked from hybridization when the samples are first flowed across the cell?

  4. I still do not get it why after sequencing the first strand and before proceeding to adding index 2 primer to generate clusters of the other strand index 1 is added again (3:17) to generate a short read. Reading the comments below has not helped. Is anyone able to explain this again? Is this done to signal to the data acquisition computer: "this is the end of sequencing data for the fragments labelled with index 1"?

  5. Hi, I have a few questions that need urgent answers:

    1/ How flourescent label in the blocking group are removed from the nucleotide?

    2/ How to wash away free nucleotide?

    3/ Why do we have to sequece 2 strain of DNA?

    4/ What is the function of index 1 and 2 in adapter attached to DNA?

    Thank you.

  6. I think the cleaving off of the fluorescent signal and removal of terminator during sequencing by synthesis is a key step that is omitted in the video. It becomes very confusing, then, how the previously added nucleotide's flourescent does not interfere with the newly added nucleotide.

  7. Amazing video with clear explanation but i have small doubt, here i found that when the reads are cleaved and washed off their respective indices are not ligated to their respective reads, how will this work in demultiplexing??

  8. Hi everybody. Here is a Sars-CoV-2 question.
    In his presentation paper of "how to make a RT-PCR test for COVID" (, the german Christian Drosten explains he sequenced the virus without any virus isolates or original patient specimens. I understand he used all the available sequences (full sequences?, part of sequences ?) of all the Cov-like viruses already known and registred in Databank, and then "aligned them". What the hell does that mean ? Did he "create" ONE sequence "gathering" all the Cov-like sequences ? A sort of "Virtual virus" ?
    And then, as far as i understand as well, he choosed three different parts of his "created alignement" supposed to be caracteristic of the "new Coronavirus" and confirmed it with the first publication of the genome by the Chines on january 11th ?
    His test method is the one recommanded by WHO and used in this pandemic from the start. As scientific and reliable is it ? Just asking.

  9. So question about experience, in terms of jobs asking to do things such as experience in PCR, NGS library prep, etc., is experience actually necessary? from being a bit exposed to it and reading it, it just seems as if you just need to follow protocols and instructions in order to do the procedures? Or can anyone elaborate on this?

  10. I contracted herpes' I was told there is no Herpes cure except treatment to control it. I totally lost hope. All I could think was losing my life because it was so embarrassing to have this virus, few weeks ago I read about a possible natural cure which was guaranteed. And I ordered the treatment after some weeks I got 100% cure. Now I'm so excited to share this testimony thanks Dr Abumere:

  11. Don't know if they meant to show these details, but the letter ordering in the forward and reverse passes is reversed but not complemented- is this an error? in 2:35 you see the ordering (from top to bottom) of AATTCGCATCG and at 3:56 on the reverse read the order is reversed but not complemented i.e. GCTACGCTTAA- shouldn't it have been complemented i.e. (C<>G,A<>T)?


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