454 sequencing

Roche 454 sequencing can sequence much longer reads than Illumina. Like Illumina, it does this by sequencing multiple reads at once by reading optical signals as bases are added.

As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case up to 1kb. Generic adaptors are added to the ends and these are annealed to beads, one DNA fragment per bead. The fragments are then amplified by PCR using adaptor-specifc primers.

Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in many PCR copies of a single sequence. The wells also contain DNA polymerase and sequencing buffers.

The slide is flooded with one of the four NTP species. Where this nucleotide is next in the sequence, it is added to the sequence read. If that single base repeats, then more will be added. So if we flood with Guanine bases, and the next in a sequence is G, one G will be added, however if the next part of the sequence is GGGG, then four Gs will be added.


The addition of each nucleotide releases a light signal. These locations of signals are detected and used to determine which beads the nucleotides are added to. 


This NTP mix is washed away. The next NTP mix is now added and the process repeated, cycling through the four NTPs.

This kind of sequencing generates graphs for each sequence read, showing the signal density for each nucleotide wash. The sequence can then be determined computationally from the signal density in each wash.

All of the sequence reads we get from 454 will be different lengths, because different numbers of bases will be added with each cycle.