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Illumina sequencing – How it works

publication date: Jun 24, 2024
 | 
author/source: INTEGRA Biosciences AG

 

llumina sequencing is a next generation sequencing method used to sequence millions of DNA fragments in parallel. In this video, INTEGRA Biosciences explain how Illumina sequencing works.

Standard Illumina sequencing can be split into the following steps:

  1. Fragmentation
  2. Addition of primer binding sites and capture sequences
  3. Denaturation
  4. Immobilization
  5. Amplification
  6. Sequencing

 

INTEGRA look at each of these steps in more detail.

  1. Fragmentation - Firstly, fragment your DNA sample into small pieces using either physical or enzymatic fragmentation methods.
  2. Addition of primer binding sites and capture sequences - Each DNA fragment needs to be elongated with adapter sequences on both ends. The inner adapter sequences are primer binding sites that are used for the sequencing reaction later on, and the outer adapter sequences are capture sequences required for immobilization.
  3. Denaturation - Once you have added the adapter sequences to your sample, denature the sequences to separate the two strands from one another.
  4. Immobilization - Your sample is added to the Illumina flow cell, which has been pre-spotted with short DNA sequences that are complementary to the capture sequences attached to your DNA sample, and consequently bind your sample sequences.
  5. Amplification - Once your sample sequences are attached to the flow cell, a single PCR cycle is performed to copy them. After this step, the sequences are denatured and the sample DNA strands are washed out. You're then left with newly synthesized strands that are immobilized to the flow cell. These immobilized sequences need to be amplified before DNA sequencing, a step that is also referred to as cluster generation. To do this, several PCR cycles are performed. The capture sequences on the flow cell are used as primers. This means that the immobilized sequences form a bridge to anneal to them during the annealing phase. The bridge is copied during the extension phase, and the two sequences separate from one another during the denaturation phase. Once there are about a thousand copies of each sequence in a cluster, one of the primers is cleaved off to ensure that you are left with only the forward or reverse strands.
  6. Sequencing - The sequencing process can now begin. A sequencing primer complementary to the primer binding site adapter on the flow cell is added. The following process is very similar to a standard PCR cycle, however, instead of dNTPs, fluorescently labeled ddNTPs are added to the reaction mixture. Unlike dNTPs, these ddNTPs lack a hydroxyl group that allows the next nucleotide to be added to it. Moreover, they are labeled with different colored fluorescent dyes for each base. As the polymerase enzyme copies the template strand, it adds the first base. As this base lacks a hydroxyl group, the reaction stops, and four images are taken – one for green, one for black, one for blue and one for red – to determine which base was added. The missing hydroxyl group can subsequently be regenerated with chemicals. These chemicals also remove the fluorescent dye from the base that was incorporated. As soon as the hydroxyl group has been regenerated, the polymerase enzyme adds the next fluorescently labeled, modified base, and the next imaging and hydroxyl group regeneration steps can take place. This process is repeated until the entire DNA strand is read out.

 

 

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