After completing theNGSsample preparation, the DNA needs to be anchored to a chip surface, where it will again be amplified and then sequenced. This chip surface is also called the flow cell (see Figure 1 below). There are numerous short DNA molecules attached to the surface of the flow cell, creating a very high-density area.

There are two short DNA sequences attached to the flow cell surface, (here in green and yellow). Each of these short DNA molecules is complementary to one strand of theadapterpreviously added to the DNA sample, thus enabling a specific binding on the flow cell. One adapter will bind to the green molecule, whereas the other adapter will bind to the yellow molecule.

Figure 1.

Cluster generation steps

After the DNA molecules are bound to the flow cell, two steps will occur. Note that the different coloring in Figure 1 and Figure 2 is irrelevant, as it is merely to show that there are two different short DNA molecules attached to the flow cell.

1.Bridge PCR

The DNA sample (in yellow) bound to the docking DNA strand (in blue) will bend and bind to a neighboring complimentary docking DNA strand molecule (in purple), creating a "bridge" (see Figure 2). A polymerase will then attach to the purple molecule and use it as a template to create a complementary copy. The bridge is then broken, and each of the DNA molecules is now only bound to one short DNA molecule. The process is repeated numerous times, generating a cluster, and in this cluster we can find DNA samples that are bound to the blue docking DNA and bound to the purple docking DNA sequence. Approximately 4,000 DNA molecules are found in each cluster after bridge PCR amplification, but half of them are washed away, leaving only 2,000 DNA molecules per cluster.

Figure 2.Cluster generation showing bridge PCR and amplification.

2.Flush

At the moment we have two complementary DNA strands attached to the flow cell surface. But we only want to sequence one of them (for example, only DNA that is bound to the blue docking DNA sequence). We will flush the other DNA molecules that are bound to the purple molecule; therefore, we will continue with DNA molecules that were synthesized with the same orientation. In each cluster, we find only DNA that is bound to the blue molecule, and they are identical because they were clonally amplified. At the end of the cluster generation step, we can expect to have approximately 200 million clonally amplified DNA clustered in one flow cell. This leaves a lot of DNA ready to be sequenced during theNGS process.