Illumina sequencing

In NGS, vast numbers of short reads are sequenced in a single stroke.

To do this, firstly the input sample must be cleaved into short sections. The length of these sections will depend on the particular sequencing machinery used.

In Illumina sequencing, 100-150bp reads are used. Somewhat longer fragments are ligated to generic adaptors and annealed to a slide using the adaptors. PCR is carried out to amplify each read, creating a spot with many copies of the same read. They are then separated into single strands to be sequenced.

The slide is flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently labelled, with the colour corresponding to the base. They also have a terminator, so that only one base is added at a time.

 

An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added.

 

The slide is then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image.

The process is repeated, adding one nucleotide at a time and imaging in between.

Computers are then used to detect the base at each site in each image and these are used to construct a sequence.

 

All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out.