3/31/2021 Illumina dye sequencing - Wikipedia

Illumina dye sequencing

Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also
known as DNA 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. It can also be used for whole-
genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery,
methylation profiling, and genome-wide protein-nucleic acid interaction analysis.[4][5]

Contents
Overview
Procedure
Genomic Library
Adapters
Bridge amplification
Clonal amplification
Sequence by synthesis
Data analysis
Comparison with other
sequencing methods
Examples of use
References

The DNA attaches to the flow cell via complementary sequences. The
Overview strand bends over and attaches to a second oligo forming a bridge. A
polymerase synthesizes the reverse strand. The two strands release and
Illumina sequencing technology straighten. Each forms a new bridge (bridge amplification). The result is a
works in three basic steps: amplify, cluster of DNA forward and reverse strands clones.
sequence, and analyze. The process
begins with purified DNA. 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. The flow cell contains nanowells that space out fragments and help with
overcrowding.[6] Each nanowell contains oligonucleotides that provide an anchoring point for the
adaptors to attach. Once the fragments have attached, a phase called cluster generation begins. This step
makes about a thousand copies of each fragment of DNA and is done by bridge amplification PCR. Next,
primers and modified nucleotides are washed onto the chip. These nucleotides have a reversible 3'
fluorescent blocker so the DNA polymerase can only add one nucleotide at a time onto the DNA
fragment.[6] After each round of synthesis, a camera takes a picture of the chip. A computer determines
what base was added by the wavelength of the fluorescent tag and records it for every spot on the chip.
After each round, non-incorporated molecules are washed away. A chemical deblocking step is then used
https://en.wikipedia.org/wiki/Illumina_dye_sequencing 1/6
3/31/2021 Illumina dye sequencing - Wikipedia

to remove the 3’ fluorescent terminal blocking group.. The process continues until the full DNA molecule
is sequenced.[5] With this technology, thousands of places throughout the genome are sequenced at once
via massive parallel sequencing.

Procedure

Genomic Library

After the DNA is purified a DNA library, genomic library, needs to be generated. There are two ways a
genomic library can be created, sonification and tagmentation. With tagmentation, transposases
randomly cuts the DNA into sizes between 50 to 500 bp fragments and adds adaptors simultaneously.[6]
A genetic library can also be generated by using sonification to fragment genomic DNA. Sonification
fragments DNA into similar sizes using ultrasonic sound waves. Right and left adapters will need to be
attached by T7 DNA Polymerase and T4 DNA ligase after sonification. Strands that fail to have adapters
ligated are washed away.[7]

Adapters

Adapters contain three different segments: the

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]
attaches to the complementary solid support.
sequence complementary to solid support
(oligonucleotides on flow cell), the barcode sequence
(indices), and the binding site for the sequencing
primer.[6] Indices are usually six base pairs long and
are used during DNA sequence analysis to identify
samples. Indices allow for up to 96 different samples
to be run together, this is also known as
multiplexing. During analysis, the computer will
group all reads with the same index together.[8][9]
Illumina uses a "sequence by synthesis" approach.[9]
This process takes place inside of an acrylamide-
coated glass flow cell.[10] The flow cell has
oligonucleotides (short nucleotide sequences)
coating the bottom of the cell, and they serve as the
solid support to hold the DNA strands in place
during sequencing. As the fragmented DNA is
washed over the flow cell, the appropriate adapter

Bridge amplification

Once attached, cluster generation can begin. The goal is to create hundreds of identical strands of DNA.
Some will be the forward strand; the rest, the reverse. This is why right and left adapters are used.
Clusters are generated through bridge amplification. DNA polymerase moves along a strand of DNA,
creating its complementary strand. The original strand is washed away, leaving only the reverse strand.
At the top of the reverse strand there is an adapter sequence. The DNA strand bends and attaches to the
oligo that is complementary to the top adapter sequence. Polymerases attach to the reverse strand, and
its complementary strand (which is identical to the original) is made. The now double stranded DNA is
denatured so that each strand can separately attach to an oligonucleotide sequence anchored to the flow
https://en.wikipedia.org/wiki/Illumina_dye_sequencing 2/6
3/31/2021 Illumina dye sequencing - Wikipedia

cell. One will be the reverse strand; the other, the forward. This
process is called bridge amplification, and it happens for
thousands of clusters all over the flow cell at once.[11]

Clonal amplification

Over and over again, DNA strands will bend and attach to the
solid support. DNA polymerase will synthesize a new strand to
create a double stranded segment, and that will be denatured so
that all of the DNA strands in one area are from a single source Millions of oligos line the bottom of
(clonal amplification). Clonal amplification is important for each flow cell lane.
quality control purposes. If a strand is found to have an odd
sequence, then scientists can check the reverse strand to make
sure that it has the complement of the same oddity. The forward and reverse strands act as checks to
guard against artefacts. 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. The reverse and forward strands should be complementary to each
other, all reverse reads should match each other, and all forward reads should match each other. If a
read is not similar enough to its counterparts (with which it should be a clone), an error may have
occurred. A minimum threshold of 97% similarity has been used in some labs' analyses.[13]

Sequence by synthesis

At the end of clonal amplification, all of the reverse strands are washed off the flow cell, leaving only
forward strands. A primer attaches to the forward strands adapter primer binding site, and a polymerase
adds a fluorescently tagged dNTP to the DNA strand. Only one base is able to be added per round due to
the fluorophore acting as a blocking group; however, the blocking group is reversible.[6] Using the four-
color chemistry, each of the four bases has a unique emission, and after each round, the machine records
which base was added. Once the color is recorded the fluorophore is washed away and another dNTP is
washed over the flow cell and the process is repeated. dATPs, dTTPs, dGTPs, and dCTPs are washed over
the cell separately so each nucleotide is able to be identified.

Starting with the launch of the NextSeq and later the MiniSeq, Illumina introduced a new two-color
sequencing chemistry. Nucleotides are distinguished by either one of two colors (red or green), no color
("black") or combining both colors (appearing orange as a mixture between red and green).

Once the DNA strand has been read, the strand that was just added is washed away. Then, the index 1
primer attaches, polymerizes the index 1 sequence, and is washed away. The strand forms a bridge again,
and the 3' end of the DNA strand attaches to an oligo on the flow cell. The index 2 primer attaches,
polymerizes the sequence, and is washed away.

A polymerase sequences the complementary strand on top of the arched strand. They separate, and the
3' end of each strand is blocked. The forward strand is washed away, and the process of sequence by
synthesis repeats for the reverse strand.

Data analysis

https://en.wikipedia.org/wiki/Illumina_dye_sequencing 3/6
3/31/2021 Illumina dye sequencing - Wikipedia

The sequencing occurs for millions of clusters at once, and each

cluster has ~1,000 identical copies of a DNA insert.[12] The sequence
data is analyzed by finding fragments with overlapping areas, called
contigs, and lining them up. If a reference sequence is known, the
contigs are then compared to it for variant identification.

This piecemeal process allows scientists to see the complete

sequence even though an unfragmented sequence was never run;
however, because Illumina read lengths are not very long[13] (HiSeq
sequencing can produce read lengths around 90 bp long[8]), it can
be a struggle to resolve short tandem repeat areas.[8][12] Also, if the
sequence is de novo and a reference doesn't exist, repeated areas can
Tagged nucleotides are added in cause a lot of difficulty in sequence assembly.[12] Additional
order to the DNA strand. Each of the difficulties include base substitutions (especially at the 3' end of
four nucleotides have an identifying reads[13]) by inaccurate polymerases, chimeric sequences, and PCR-
label that can be excited to emit a bias, all of which can contribute to generating an incorrect
characteristic wavelength. A sequence.[13]
computer records all of the
emissions, and from this data, base
calls are made. Comparison with other sequencing
methods
This technique offers several advantages over traditional sequencing methods such as Sanger
sequencing. Sanger sequencing requires two reactions, one for the forward primer and another for the
reverse primer. Unlike Illumina, Sanger sequencing uses fluorescently labeled dideoxynucleoside
triphosphates (ddNTPs) to determine the sequence of the DNA fragment. ddNTPs are missing the 3' OH
group and terminates DNA synthesis permanently.[6] In each reaction tube, dNTPs and ddNTPs are
added, along with DNA polymerase and primers. The ratio of ddNTPs to dNTPs matter since the
template DNA needs to be completely synthesized, and an overabundance of ddNTPs will create multiple
fragments of the same size and position of the DNA template. When the DNA polymerase adds a ddNTP
the fragment is terminated and a new fragment is synthesized. Each fragment synthesized is one
nucleotide longer than the last. Once the DNA template has been completely synthesized, the fragments
are separated by capillary electrophoresis. At the bottom of the capillary tube a laser excites the
fluorescently labeled ddNTPs and a camera captures the color emitted.

Due to the automated nature of Illumina dye sequencing it is possible to sequence multiple strands at
once and gain actual sequencing data quickly. With Sanger sequencing, only one strand is able to be
sequenced at a time and is relatively slow. Illumina only uses DNA polymerase as opposed to multiple,
expensive enzymes required by other sequencing techniques (i.e. pyrosequencing).[14]

Examples of use
Illumina sequencing has been used to research transcriptomes of the sweet potato[15] and the
gymnosperm genus Taxus.[16]

References
1. CA 2158975 (http://www.google.ge/patents/CA2158975A1), Canard B, Sarfati S, "Novel derivatives
usable for the sequencing of nucleic acids", published 13 October 1994, assigned to Pasteur
Institute
https://en.wikipedia.org/wiki/Illumina_dye_sequencing 4/6