Illumina dye sequencing

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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

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. Cluster Generation.png
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.

Overview

This works in three basic steps: amplify, 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 adapters 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 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 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: sonication and tagmentation. With tagmentation, transposases randomly cuts the DNA into sizes between 50 and 500 bp fragments and adds adaptors simultaneously. [6] A genetic library can also be generated by using sonication to fragment genomic DNA. Sonication 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 sonication. Strands that fail to have adapters ligated are washed away. [7]

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 DNA Processing Preparation.png
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

Adapters

Adapters contain three different segments: the 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 attaches to the complementary solid support.

Millions of oligos line the bottom of each flow cell lane. Oligonucleotide chains in Flow Cell.png
Millions of oligos line the bottom of each flow cell lane.

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 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 (clonal amplification). Clonal amplification is important for 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.

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).

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. Sequence By Synthesis.png
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.

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

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 does not exist, repeated areas can cause a lot of difficulty in sequence assembly. [12] Additional difficulties include base substitutions (especially at the 3' end of reads [13] ) by inaccurate polymerases, chimeric sequences, and PCR-bias, all of which can contribute to generating an incorrect sequence. [13]

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]

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<span class="mw-page-title-main">Complementary DNA</span> Single-stranded DNA synthesized from RNA

In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a specific protein in a cell that does not normally express that protein, or to sequence or quantify mRNA molecules using DNA based methods. cDNA that codes for a specific protein can be transferred to a recipient cell for expression, often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays, qPCR, and RNA-seq.

<span class="mw-page-title-main">Primer (molecular biology)</span> Short strand of RNA or DNA that serves as a starting point for DNA synthesis

A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.

In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.

<span class="mw-page-title-main">DNA sequencer</span> A scientific instrument used to automate the DNA sequencing process

A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine). This is then reported as a text string, called a read. Some DNA sequencers can be also considered optical instruments as they analyze light signals originating from fluorochromes attached to nucleotides.

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

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<span class="mw-page-title-main">Sanger sequencing</span> Method of DNA sequencing developed in 1977

Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. It was first commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger sequencing has been replaced by next generation sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still has the advantage over short-read sequencing technologies in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%. Sanger sequencing is still actively being used in efforts for public health initiatives such as sequencing the spike protein from SARS-CoV-2 as well as for the surveillance of norovirus outbreaks through the Center for Disease Control and Prevention's (CDC) CaliciNet surveillance network.

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<span class="mw-page-title-main">ABI Solid Sequencing</span>

SOLiD (Sequencing by Oligonucleotide Ligation and Detection) is a next-generation DNA sequencing technology developed by Life Technologies and has been commercially available since 2006. This next generation technology generates 108 - 109 small sequence reads at one time. It uses 2 base encoding to decode the raw data generated by the sequencing platform into sequence data.

<span class="mw-page-title-main">2 base encoding</span>

2 Base Encoding, also called SOLiD, is a next-generation sequencing technology developed by Applied Biosystems and has been commercially available since 2008. These technologies generate hundreds of thousands of small sequence reads at one time. Well-known examples of such DNA sequencing methods include 454 pyrosequencing, the Solexa system and the SOLiD system. These methods have reduced the cost from $0.01/base in 2004 to nearly $0.0001/base in 2006 and increased the sequencing capacity from 1,000,000 bases/machine/day in 2004 to more than 100,000,000 bases/machine/day in 2006.

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<span class="mw-page-title-main">Reduced representation bisulfite sequencing</span> Methylation process

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<span class="mw-page-title-main">Duplex sequencing</span>

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References

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  2. ↑ Canard B, Sarfati RS (October 1994). "DNA polymerase fluorescent substrates with reversible 3'-tags". Gene. 148 (1): 1–6. doi:10.1016/0378-1119(94)90226-7. PMID   7523248.
  3. ↑ "History of Illumina Sequencing". Archived from the original on 12 October 2014.
  4. ↑ "Illumina - Sequencing and array-based solutions for genetic research". www.illumina.com.
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  14. ↑ Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (1998-07-17). "A Sequencing Method Based on Real-Time Pyrophosphate". Science. 281 (5375): 363–365. doi:10.1126/science.281.5375.363. ISSN   0036-8075. PMID   9705713.
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