After thecluster generation, we are ready to begin the sequencing process. There are several different ways of performingNext Generation Sequencing, depending on the platform that you are using. In this lab, we are using Illumina’ssequencing by synthesis. This implies that the sequencing data is obtained by synthesizing each base pair. Let us go through the process in detail.

Polymerase attachment

Once we have the cluster of clonal DNA attached to the flow cell, sequencing reagents are pumped into it. They enter from one side of the flow cell and exit on the other side. T4 DNA polymerase is flushed in and attaches to the short DNA molecules bound to the flow cell. The T4 polymerase is modified to only allow incorporation of one base.

All the 4 nucleotides are tagged with a specific fluorophore. As you can see in Figure 1, Thymine is tagged with agreenfluorophore, Cytosine with abluefluorophore, Guanine with aredfluorophore, and Adenine with anorangefluorophore. These nucleotides are also modified: they have a 3' cap that allows only 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. Any remaining free-floating nucleotides are then washed away from the system.

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, the next sequencing cycle to begin.Nucleotides tagged with fluorophore

Detection

Because all the nucleotides are individually labeled with a specific fluorophore, we can identify the nucleotides by observing the fluorescence signal that they emit. For example, in Figure 1 we can observe that one nucleotide tagged with agreenfluorophore has been added at the 3' end of the primer. A chemical reaction triggers the emission of thegreenlight 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 pictures are taken at the end of each cycle, recording each of the possible colors. After taking all four pictures, the system will overlay them. Therefore, we can easily identify the base identity of each cluster (see Figure 2).

Figure 2.An overlay image, each color dot represents a clonally amplified DNA cluster. The color codes for the four different nucleotides: Thymine, Cytosine, Guanine, and Adenine.

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 again.

After sequencing, the images are fed into a computer and thesequencing data can be analyzedwith an specific software.