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Single- vs. Paired-end Sequencing

Single-end sequencing

As the name suggests, single-end sequencing is when the sequencing process is performed only from a single direction. As we have discussed earlier in cluster generation after bridge PCR amplification, we have two types of DNA molecules that are complementary to each other, and we washed away one of them. We then sequence the remaining DNA that has the same direction. If we only sequence this DNA (all having the same direction), it is called by single-end sequencing.

Paired-end sequencing

In paired-end sequencing, the DNA is being sequenced from both directions (see Figure 3). The process is identical to the single-end sequencing, but after the end of the sequencing process, we continue by running another round of cluster generation and removing the strands with the direction that we have sequenced previously (the DNA having blue adapters). Therefore, we are left with only DNA having purple adapters. This DNA has the complementary sequence of the first DNA.

When using paired-end sequencing, we can detect structural variants in the genome with higher confidence because we have higher sequence coverage and increased specificity when aligning back to the genome compared with single-end sequencing, which involves only one end of the DNA fragment. When sequencing long DNA fragments, it is preferred to use paired-end sequencing. It is also better to use paired-end sequencing when we want to study deletion, mutations, or chromosomal rearrangements.

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 substrate. 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. The original DNA strand is then removed.

Figure 1. Paired-end sequencing implies that the sequencing is performed from both directions. The DNA needs to be flipped by running another round of cluster generation.

Referred from:

Article
Sequencing process