Question

Why is the cloning step unnecessary prior to sequencing with massively parallel sequencing (MPS; Illumina or similar technologies), when it is necessary for hierarchical and shotgun sequencing?

I know that the cloning step is helpful for introducing universal primer binding sites for sequencing. So is it because we are using probes with adaptors on them for MPS that we don't need to clone?

Answer 1

Cloning is unnecessary for massively parallel sequencing techniques due to the direct amplification and sequencing of DNA fragments with adaptors containing primer binding sites, leading to significant improvements in sequencing efficiency.

Step-by-step explanation:

The cloning step is unnecessary in massively parallel sequencing technologies such as Illumina's because these methodologies are capable of amplifying and sequencing DNA fragments directly without the need to insert them into a cloning vector. In contrast, traditional hierarchical and shotgun sequencing required cloning because they dealt with larger fragments that needed to be individually isolated and amplified before sequencing.

In massively parallel sequencing, adaptors with universal primer binding sites are attached to the DNA fragments, which are then immobilized and amplified on a surface, allowing for many fragments to be sequenced simultaneously. This represents a significant improvement in efficiency and throughput when compared with previous methods like shotgun sequencing, where the cloning step was critical for amplifying DNA fragments to obtain an adequate signal for sequencing.