Illumina dye sequencing

The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris.

[1][2] It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University,[3] who subsequently founded Solexa, a company later acquired by Illumina.

This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands.

The DNA is fragmented and adapters are added that contain segments that act as reference points during amplification, sequencing, and analysis.

The modified DNA is loaded onto a flow cell where amplification and sequencing will take place.

This step makes about a thousand copies of each fragment of DNA and is done by bridge amplification PCR.

A computer determines what base was added by the wavelength of the fluorescent tag and records it for every spot on the chip.

A chemical deblocking step is then used to remove the 3’ fluorescent terminal blocking group.

With tagmentation, transposases randomly cut the DNA into sizes between 50 and 500 bp fragments and add adaptors simultaneously.

[6] Indices are usually six base pairs long and are used during DNA sequence analysis to identify samples.

As the fragmented DNA is washed over the flow cell, the appropriate adapter attaches to the complementary solid support.

The DNA strand bends and attaches to the oligo that is complementary to the top adapter sequence.

Because Illumina sequencing uses DNA polymerase, base substitution errors have been observed,[12] especially at the 3' end.

[13] Paired end reads combined with cluster generation can confirm an error took place.

Starting with the launch of the NextSeq and later the MiniSeq, Illumina introduced a new two-color sequencing chemistry.

[12] The sequence data is analyzed by finding fragments with overlapping areas, called contigs, and lining them up.

At the bottom of the capillary tube a laser excites the fluorescently labeled ddNTPs and a camera captures the color emitted.

Illumina only uses DNA polymerase as opposed to multiple, expensive enzymes required by other sequencing techniques (i.e.

The DNA attaches to the flow cell via complementary sequences. The strand bends over and attaches to a second oligo forming a bridge. A polymerase synthesizes the reverse strand. The two strands release and straighten. Each forms a new bridge (bridge amplification). The result is a cluster of DNA forward and reverse strand clones.
Double stranded DNA is cleaved by transposomes. The cut ends are repaired and adapters, indices, primer binding sites, and terminal sites are added to each strand of the DNA. Image based in part on illumina's sequencing video [ 7 ]
Millions of oligos line the bottom of each flow cell lane.
Tagged nucleotides are added in order to the DNA strand. Each of the four nucleotides have an identifying label that can be excited to emit a characteristic wavelength. A computer records all of the emissions, and from this data, base calls are made.