Sequencing process

Now that we have a flow cell filled with a lot of DNA, we are ready to begin the sequencing process. There are several different ways of performing Next Generation Sequencing depending on the platform that you are using. One of them is sequencing by synthesis, in which the sequencing data is obtained by synthesizing each base pair.

Figure 1. Sequencing by synthesis. After the polymerase has attached to the primer, nucleotides labeled with a specific fluorophore will be added at the 3' end of the primer. The nucleotides are modified with a 3' cap that allows only one nucleotide addition at a time. The fluorescence signal emitted by the fluorophore is detected, and the 3' cap is cleaved, allowing for the next sequencing cycle to begin.

Polymerase attachment

Once we have the cluster of clonal DNA attaching to the flow cell, sequencing reagents are then pumped into the flow cell. They enter on one side of the flow cell and exit on the other side. T4 DNA polymerase is flushed in and attaches to the short DNA molecule bound to the flow cell (acting as the sequencing primer).

Nucleotides tagged with fluorophore

Each of the four nucleotides are tagged with a specific fluorophore. For example, thymine is tagged with a green fluorophore, cytosine with a blue fluorophore, guanine with a red fluorophore, and adenine with an orange fluorophore. These nucleotides are also modified; they have a 3' cap that only allows for the addition of one nucleotide at a time. Therefore, after one nucleotide has been added to the 3' end of the primer, no more nucleotides can attach. The remaining nucleotides that are freely floating are then washed away from the system.

Fluorophore Detection

Because all of the nucleotides are individually labelled with a specific fluorophore, we can identify the nucleotides by observing the fluorescence signal that they emit. For example, in Figure 1, we can see that one nucleotide tagged with a green fluorophore is added at to the 3' end of the primer. A chemical reaction triggers the emission of the green light that can be recorded by taking a picture. Because we know that thymine is tagged with the green fluorophore, we can identify the base as thymine. Four photos are taken at the end of each cycle, recording each of the possible colors. After taking all four pictures, the system will overlay all four images; therefore we can quickly identify the base identity of each cluster.

Cleavage and removal

After the picture has been taken the 3' cap of the nucleotide is cleaved off so that the next sequencing cycle can begin. The cleaved 3' cap is then removed from the system. This marks the end of a sequencing cycle and the new cycle can begin by flushing new rounds of nucleotides and repeating the steps.

For more information: - Single vs Paired-end Sequencing