High Throughput Sequencing
High Throughput Sequencing (also known as Illumina Solexa DNA sequencing) is a technique used to sequence DNA or cDNA via fragmenting the genetic information randomly. RNA can also be sequenced but involves additional steps to those shown below. Firstly mRNA is hydrolysed by magnesium catalysed reaction to give fragments and then the sequences are randomly primed by reverse transcription .
Double stranded DNA (dsDNA) is randomly fragmented to blund ended pieces. Attach adapters to either end of the fragmented dsDNA via blunt end ligation. Two adapters are added separately so both ends of the dsDNA have an adapter present.
Bridge Amplification- The free end of the fragment bends and hybridises to a separate complimentary primer on the flow cell forming a 'bridge'. Unlabelled nucleotides and enzymes are then added to the flow cell to initiate amplification of the second strand of DNA.
First Chemistry Cycle- Add four reversible terminators, primers and DNA polymerase to the flow cell. Each cluster will show up as spot after laser excitation due to the added bases fluorescence. The blocked 3' OH terminus and the flurophone from each base are then removed to allow another base to be added to the sequence. This will eventually allow the full sequence to be built up.
- ↑ Mardis ER. (2008), "Next-generation DNA sequencing methods", Annu Rev Genomics Hum Genet.,9:387-402
Mardis ER (2008) Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet. 9:387-402