Labster Logo

Cluster generation

After completing the NGS sample preparation, the DNA needs to be anchored to a plate surface where it will again be amplified and then sequenced. This plate surface is also called a flow cell and has a high density of short DNA strands attached to its surface. The DNA molecules are able to bind to an adapter on the flow cell, as the sequences are complementary.

CLUSTER GENERATION STEPS:

After the DNA molecules are bound to the flow cell, two steps will occur.

Bridge PCR

The DNA molecule that is bound to the short DNA molecule (shown as the blue molecule) will bend over and bind to the other molecule (shown as the purple molecule), creating a "bridge" (see Figure 1). A polymerase will then attach to the purple molecule and amplify the DNA, creating two DNA molecules that are complementary to each other. The bridge is then released, 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 both the DNAs, the DNA molecules that are bound to the blue molecule and the DNA molecule that is attached to the purple molecule. 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. This step is described below.

Flush

At the moment we have two complementary DNA strands attaching to the flow cell surface. But we only want to sequence clusters of identical (clonal) DNA (for example, only DNA that is bound to the blue molecule). We will flush the other DNA molecules that are linked to the purple molecule; therefore, now we will have only DNA molecules that were synthesized with the same orientation. Now, in each cluster, we only find 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 cluster in one flow cell. This is a lot of DNA that is ready to be sequenced!

In step 1, a strand of DNA is vertical and has an adapter on each end, one colored blue and one colored purple. The blue adapter is attached to the flow cell which has a high density of short DNA strands attached to its surface. In step 2, the strand of DNA bends over so both adapters are pointing at the substrate. In step 3, the strand of DNA is copied to make a new DNA strand that is also bent over. The copying starts from the original purple adapter, and the new purple adapter is attached to the substrate. In step 4, both strands are now vertical, but the copied DNA strand is attached to the substrate with the purple adapter, and the original DNA strand is attached to the substrate with the blue adapter. This copying repeats itself multiple times until many DNA strands have been made.

Figure 1. Cluster generation is showing bridge PCR and amplification.

Referred from:

Article
Next Generation Sequencing
Article
Adapter ligation
Article
Single- vs. Paired-end Sequencing
Article
Welcome to Next Generation Sequencing (NGS) Lab