Everything about Genome Sequencing totally explained
The term
DNA sequencing encompasses
biochemical methods for determining the order of the
nucleotide bases,
adenine,
guanine,
cytosine, and
thymine, in a
DNA oligonucleotide. The sequence of DNA constitutes the
heritable genetic information in
nuclei,
plasmids,
mitochondria, and
chloroplasts that forms the basis for the developmental programs of all living organisms. Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or
forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the large-scale sequencing of the
human genome, in the
Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.
Early methods
For thirty years, a large proportion of DNA sequencing has been carried out with the chain-termination method developed by
Frederick Sanger and coworkers in 1975. Prior to the development of rapid DNA sequencing methods in the early 1970s by Sanger in England and
Walter Gilbert and
Allan Maxam at
Harvard, a number of laborious methods were used. For instance, in 1973 Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis.
RNA sequencing, which for technical reasons is easier to perform than DNA sequencing, was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing, dating from the pre-recombinant DNA era, is the sequence of the first complete gene and then the complete genome of
Bacteriophage MS2, identified and published by
Walter Fiers and his coworkers at the
University of Ghent (
Ghent,
Belgium), published between 1972 and 1976..
Maxam-Gilbert sequencing
In 1976-1977,
Allan Maxam and
Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases
(External Link
).
Although Maxam and Gilbert published their chemical sequencing method two years after the ground-breaking paper of Sanger and Coulson on plus-minus sequencing, Maxam-Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. However, with the development and improvement of the chain-termination method (see below), Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity, extensive use of hazardous chemicals, and difficulties with scale-up. In addition, unlike the chain-termination method, chemicals used in the Maxam-Gilbert method can't easily be customized for use in a standard molecular biology kit.
In brief, the method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments are then size-separated by
gel electrophoresis, with the four reactions arranged side by side. To visualize the fragments generated in each reaction, the gel is exposed to
X-ray film for
autoradiography, yielding an image of a series of dark 'bands' corresponding to the
radiolabelled DNA fragments, from which the sequence may be inferred.
Also sometimes known as 'chemical sequencing', this method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.
Chain-termination methods
While the chemical sequencing method of Maxam and Gilbert, and the plus-minus method of Sanger and Coulson were orders of magnitude faster than previous methods, the chain-terminator method developed by Sanger was even more efficient, and rapidly became the method of choice. The Maxam-Gilbert technique requires the use of highly toxic chemicals, and large amounts of
radiolabeled DNA, whereas the chain-terminator method uses fewer toxic chemicals and lower amounts of radioactivity. The key principle of the Sanger method was the use of
dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators.
The classical chain-termination or Sanger method requires a single-stranded DNA template, a DNA
primer, a
DNA polymerase, radioactively or
fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing the four standard
deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the
DNA polymerase. To each reaction is added only one of the four
dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). These dideoxynucleotides are the chain-terminating nucleotides, lacking a 3'-
OH group required for the formation of a
phosphodiester bond between two nucleotides during DNA strand elongation. Incorporation of a dideoxynucleotide into the nascent (elongating) DNA strand therefore terminates DNA strand extension, resulting in various DNA fragments of varying length. The dideoxynucleotides are added at lower concentration than the standard deoxynucleotides to allow strand elongation sufficient for sequence analysis.
The newly synthesized and labeled DNA fragments are heat
denatured, and separated by size (with a resolution of just one nucleotide) by
gel electrophoresis on a denaturing
polyacrylamide-urea gel. Each of the four DNA synthesis reactions is run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by
autoradiography or UV light, and the DNA sequence can be directly read off the
X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that's the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The terminal nucleotide base can be identified according to which dideoxynucleotide was added in the reaction giving that band. The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence as indicated.
There are some technical variations of chain-termination sequencing. In one method, the DNA fragments are tagged with nucleotides containing radioactive phosphorus for
radiolabelling. Alternatively, a primer labeled at the 5’ end with a
fluorescent dye is used for the tagging. Four separate reactions are still required, but DNA fragments with dye labels can be read using an optical system, facilitating faster and more economical analysis and automation. This approach is known as 'dye-primer sequencing'. The later development by L Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
The different chain-termination methods have greatly simplified the amount of work and planning needed for DNA sequencing. For example, the chain-termination-based "Sequenase" kit from
USB Biochemicals contains most of the reagents needed for sequencing, prealiquoted and ready to use. Some sequencing problems can occur with the Sanger method, such as non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence. In addition, secondary structures within the DNA template, or contaminating RNA randomly priming at the DNA template can also affect the fidelity of the obtained sequence. Other contaminants affecting the reaction may consist of extraneous DNA or inhibitors of the DNA polymerase.
Dye-terminator sequencing
An alternative to primer labelling is labelling of the chain terminators, a method commonly called 'dye-terminator sequencing'. The major advantage of this method is that the sequencing can be performed in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with a different fluorescent dye, each fluorescing at a different
wavelength. This method is attractive because of its greater expediency and speed and is now the mainstay in automated sequencing with computer-controlled sequence analyzers (see below). Its potential limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace
chromatogram after
capillary electrophoresis (see figure to the right). This problem has largely been overcome with the introduction of new DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs", caused by certain chemical characteristics of the dyes that can result in artifacts in DNA sequence traces. The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects, as it's both easier to perform and lower in cost than most previous sequencing methods.
Challenges
Modern sequencing typically produces a sequence that has poor quality in the first 15-40 bases, a high quality region of 700-900 bases, and then quickly deteriorating quality.
Base calling software typically outputs an estimate of quality along with the sequence to aid in
quality trimming.
Before the DNA can be sequenced,
linker sequences are attached to its ends, and it's inserted into a
cloning vector. The resulting sequence can therefore often contain parts of the vector or the linker sequences, which must be filtered out prior to analysis. In contrast, emerging sequencing technologies based on
pyrosequencing often avoid using cloning vectors.
During
PCR amplification, unrelated sequences can hybridize, and the resulting clone can be a
chimaeric sequence, containing fragments from both sequences. Another problem is
polymerase stuttering, where the
polymerase repeatedly outputs the same fragments, giving an artificially long low-complexity part of the sequence.
Automation and sample preparation
Modern automated DNA sequencing instruments (
DNA sequencers) can sequence up to 384 fluorescently labelled samples in a single batch (run) and perform as many as 24 runs a day. However, automated DNA sequencers carry out only DNA-size-based separation (by
capillary electrophoresis), detection and recording of dye fluorescence, and data output as fluorescent peak trace
chromatograms. Sequencing reactions by
thermocycling, cleanup and re-suspension in a
buffer solution before loading onto the sequencer are performed separately. In the past, an operator had to trim the low quality ends (see image in the right) of every sequence manually in order to remove the sequencing errors. However, today, software like
Fast Chromatogram Viewer can automatically trim the ends at batch.
Large-scale sequencing strategies
Current methods can directly sequence only relatively short (300-1000
nucleotides long) DNA fragments in a single reaction.
(External Link). The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide. Limitations on ddNTP incorporation were largely solved by Tabor at Harvard Medical, Carl Fuller at USB biochemicals, and their coworkers
The human genome is about 3 billion (3,000,000,000) bp long; if the average fragment length is 500 bases, it would take a minimum of six million (3 billion/500) to sequence the human genome (not allowing for overlap = 1-fold coverage). Keeping track of such a high number of sequences presents significant challenges, only held down by developing and coordinating several procedural and computational
algorithms, such as efficient database development and management.
Resequencing or
targeted sequencing is utilized for determining a change in DNA sequence from a "reference" sequence. It is often performed using PCR to amplify the region of interest (pre-existing DNA sequence is required to design the PCR primers). Resequencing uses three steps, extraction of DNA or RNA from biological tissue; amplification of the RNA or DNA (often by PCR); followed by sequencing. The resultant sequence is compared to a reference or a normal sample to detect mutations.
New sequencing methods
High-throughput sequencing
The high demand for low cost sequencing has given rise to a number of high-throughput sequencing technologies. These efforts have been funded by public and private institutions as well as privately researched and commercialized by biotechnology companies. High-throughput sequencing technologies are intended to lower the cost of sequencing DNA libraries beyond what is possible with the current dye-terminator method based on DNA separation by capillary electrophoresis. Many of the new high-throughput methods use methods that parallelize the sequencing process, producing thousands or millions of sequences at once.
In vitro clonal amplification
As molecular detection methods are often not sensitive enough for single molecule sequencing, most approaches use an in vitro cloning step to generate many copies of each individual molecule. Emulsion PCR is one method, isolating individual DNA molecules along with primer-coated beads in aqueous bubbles within an oil phase. A polymerase chain reaction (PCR) then coats each bead with clonal copies of the isolated library molecule and these beads are subsequently immobilized for later sequencing. Emulsion PCR is used in the methods published by Marguilis et al. (commercialized by 454 Life Sciences, acquired by Roche), Shendure and Porreca et al. (also known as "polony sequencing") and SOLiD sequencing, (developed by Agencourt and acquired by Applied Biosystems). Another method for in vitro clonal amplification is "bridge PCR", where fragments are amplified upon primers attached to a solid surface, developed and used by Solexa (now owned by Illumina). These methods both produce many physically isolated locations which each contain many copies of a single fragment. The single-molecule method developed by Stephen Quake's laboratory (later commercialized by Helicos) skips this amplification step, directly fixing DNA molecules to a surface.
Parallelized sequencing
Once clonal DNA sequences are physically localized to separate positions on a surface, various sequencing approaches may be used to determine the DNA sequences of all locations, in parallel. "Sequencing by synthesis", like the popular dye-termination electrophoretic sequencing, uses the process of DNA synthesis by DNA polymerase to identify the bases present in the complementary DNA molecule. Reversible terminator methods (used by Illumina and Helicos) use reversible versions of dye-terminators, adding one nucleotide at a time, detecting fluorescence corresponding to that position, then removing the blocking group to allow the polymerization of another nucleotide. Pyrosequencing (used by 454) also uses DNA polymerization to add nucleotides, adding one type of nucleotide at a time, then detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates.
"Sequencing by ligation" is another enzymatic method of sequencing, using a DNA ligase enzyme rather than polymerase to identify the target sequence. Mass spectrometry can also be used to sequence DNA molecules; conventional chain-termination reactions produce DNA molecules of different lengths and the length of these fragments is then determined by the mass differences between them (rather than using gel separation).
There are new proposals for DNA sequencing, which are in development, but remain to be proven. These include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores, and microscopy-based techniques, such as AFM or electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments by nucleotide labeling with heavier elements (for example, halogens) for visual detection and recording. In October 2006 the NIH issued a news release describing novel sequencing techniques and announcing several grant awards.
In October 2006, the X Prize Foundation established the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome."
Major landmarks in DNA sequencing
1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA.
1975 The first complete DNA genome to be sequenced is that of bacteriophage φX174
1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation" (External Link). Fred Sanger, independently, publishes "DNA sequencing by enzymatic synthesis".
1980 Fred Sanger and Wally Gilbert receive the Nobel Prize in Chemistry
1982 Genbank starts as a public repository of DNA sequences.
1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb.
1985 Kary Mullis and colleagues develop the polymerase chain reaction, a technique to replicate small fragments of DNA
1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing machine.
1987 Applied Biosystems markets first automated sequencing machine, the model ABI 370.
1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at 75 cents (US)/base).
1991 Craig Venter develops strategy to find expressed genes with ESTs (Expressed Sequence Tags).
- Uberbacher develops GRAIL, a gene-prediction program.
1992 Craig Venter leaves NIH to set up The Institute for Genomic Research (TIGR).
- William Haseltine heads Human Genome Sciences, to commercialize TIGR products.
- Wellcome Trust begins participation in the Human Genome Project.
- Simon et al. develop BACs (Bacterial Artificial Chromosomes) for cloning.
- First chromosome physical maps published:
- Page et al. - Y chromosome;
- Cohen et al. chromosome 21.
- Lander - complete mouse genetic map;
- Weissenbach - complete human genetic map.
1993 Wellcome Trust and MRC open Sanger Centre, near Cambridge, UK.
- The GenBank database migrates from Los Alamos (DOE) to NCBI (NIH).
1995 Venter, Fraser and Smith publish first sequence of free-living organism, Haemophilus influenzae (genome size of 1.8 Mb).
- Richard Mathies et al. publish on sequencing dyes (PNAS, May).
- Michael Reeve and Carl Fuller, thermostable polymerase for sequencing.
1996 International HGP partners agree to release sequence data into public databases within 24 hours.
- International consortium releases genome sequence of yeast S. cerevisiae (genome size of 12.1 Mb).
- Yoshihide Hayashizaki's at RIKEN completes the first set of full-length mouse cDNAs.
- ABI introduces a capillary electrophoresis system, the ABI310 sequence analyzer.
1997 Blattner, Plunkett et al. publish the sequence of E. coli (genome size of 5 Mb)
1998 Phil Green and Brent Ewing of Washington University publish “phred” for interpreting sequencer data (in use since ‘95).
- Venter starts new company “Celera”; “will sequence HG in 3 yrs for $300m.”
- Applied Biosystems introduces the 3700 capillary sequencing machine.
- Wellcome Trust doubles support for the HGP to $330 million for 1/3 of the sequencing.
- NIH & DOE goal: "working draft" of the human genome by 2001.
- Sulston, Waterston et al finish sequence of C. elegans (genome size of 97Mb).
1999 NIH moves up completion date for rough draft, to spring 2000.
- NIH launches the mouse genome sequencing project.
- First sequence of human chromosome 22 published.
2000 Celera and collaborators sequence fruit fly Drosophila melanogaster (genome size of 180Mb) - validation of Venter's shotgun method. HGP and Celera debate issues related to data release.
- HGP consortium publishes sequence of chromosome 21.
- HGP & Celera jointly announce working drafts of HG sequence, promise joint publication.
- Estimates for the number of genes in the human genome range from 35,000 to 120,000. International consortium completes first plant sequence, Arabidopsis thaliana (genome size of 125 Mb).
2001 HGP consortium publishes Human Genome Sequence draft in Nature (15 Feb).
- Celera publishes the Human Genome sequence.
2005 420,000 VariantSEQr human resequencing primer sequences published on new NCBI Probe database.
2007 For the first time, a set of closely related species (12 Drosophilidae) are sequenced, launching the era of phylogenomics.
- Craig Venter publishes his full diploid genome: the first human genome to be sequenced completely.
2008 An international consortium launches The 1000 Genomes Project, aimed to study human genetic variability.Further Information
Get more info on 'Genome Sequencing'.
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