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Single molecule sequencing first made its debut in 2003 and is based on the principle of asynchronous synthesis. Aside from sequencing genomes, asynchrony could also be used to assist with the reading of base repeat sequences and homopolymers. Single molecule sequencing capitalizes on the fact that this technology does not require genomic DNA to be cloned, amplified, or ligated before it is sequenced. This will enable the cost and time required for genomic sequencing to be made more affordable, while providing high throughput sequencing technology.
Studies conducted by Pushkarev et al., 2009, demonstrated that single molecule sequencing was very accurate. Pushkarev et al., 2009 sequenced a complete human genome, approximately 3.8million base pairs, using single molecule sequencing and found that the results obtained had a false-positive rate of less than 1%. The low rate of mistakes was possible as each template molecule was examined separately from other template molecules. This provided the advantage of low rate of mis-incorporation of the wrong base as the kinetics of base incorporation do not compete with the kinetics of template molecule reading. Single molecule sequencing also allows template molecules to be sequenced in situ, which would also reduce the rate of misincorporation of bases.
Helicos Bioscience was the first research company to release a platform, Heliscope Single Molecule Sequencing, which enabled genome to be sequence using sequencing-by-synthesis procedures. Heliscope Single Molecule Sequencing enables up to 1 billion base pairs to be sequenced within a week. This would enable rapid and large scale sequencing of genomes, including sequencing of the human genome, which was demonstated by Pushkarev et al., 2009. Heliscope Single Molecule Sequencing operates by sequencing individual nucleic acid molecules in DNA or RNA which were first melted into single stranded DNA and poly-A tail. The single stranded DNA are then placed onto the surface of the glass Heliscope Flow Cell which was coated with oligonucleotide. The Heliscope Flow Cell is then filled with dTTP and polymerase in order to fill any remaining nucleotides which are complementary to the poly A-tail. Then, fluorescently labeled nucleotide is added and sequencing is carried out. Fluorescently labeled nucleotide which is complementary to the template DNA is detected by emission of fluorescent light once laser is shown through the flow-cell surface. Heliscope then captures the image and records the position of the fluorescent nucleotides, following which the fluorescence will be cleaved from the incorporated nucleotides. The cycle is then repeated, with Heliscope Single Molecule Sequencing continually adding in complementary nucleotides in following run cycles.
Although single molecule sequencing has many advantages, single molecule sequencing does have its own setbacks. It was found that when SNPs were being sequenced, presence of nonzero false-positive and false-negative rates were high.
Another advanced sequencing technology which has gained popularity is real time single molecule DNA sequencing. DNA polymerase was chosen as the method used for real time single molecule sequencing as DNA polymerase is a stable, single-subunit enzyme that has a rapid, efficient, high processivity rate for duplicating genomes. As DNA polymerases have proofreading activity, the error rates exhibited were very low, approximately one in every 105 bases.
An advantage of using DNA polymerase was that DNA polymerase would be able to sequence close circular templates multiple times in a single run. This would reduce the cost of sequencing the genome as only one DNA molecule was needed to determine a circular consensus sequence. Reagent consumption would also be minimized as only small amounts of genomic DNA are required for a single run. Real time single molecule sequencing would also decrease the amount of time needed to sequence a genome by about four orders of magnitude compared to Sanger sequencing. This was because the real time single molecule sequencing enables thousands of bases to be read simultaneously due to continuous DNA synthesis.
The platform used for real time single molecule real time sequencing was developed by Pacific Biosciences. The instrument is a nanophotonic structure which uses high-multiplex confocal fluorescence detection system that targets uniform multilaser illumination of 3000 zero-mode waveguide (ZMWs) through holographic phase masks. This instrument uses a confocal pinhole array to reject out-of-focus background, and a prism dispersive element for wavelength discrimination. This would enable rapid and accurate sequencing while providing flexibility in the choice of fluorescent dyes used for transmitting up to 99% of the incident light.
It was found that if genome was sequenced using real time single molecule sequencing, errors were detected due to deletions which occurred due to unlabeled nucleotide contamination. This error could be overcomed by using a dNTP composition which was more than 99.5% pure. Another method would be to reduce the fraction of short incorporations events in the genome, increase fluorophore brightness and improve efficiency of light collection of the detection system.
Studies conducted by Eid et al., 2009, showed a 99.3% accuracy of the genome sequenced. In order to increase the accuracy of the genome being sequenced, different dyes and absorption wavelengths could be used. Dyes which provide larger spectral separations while at the same time increasing the brightness of the instrument would provide higher accuracy of the genome being sequenced. The merged of rapid single molecule sequencing and real time DNA sequencing enabled rapid, large scale and cost-effective sequencing of genomes. This would allow independent laboratories to carry out research which was previously only carried out by major genome institutes.
Hybridization sequencing sequences DNA based on the principle that different oligonucleotides will hybridize on different probes. The advantage of using hybridization sequencing is that a large amount of sequences could be obtained from the genome in a relatively short amount of time. This was because hybridization sequencing uses microarrays, which are able to read up to 109 base pairs. The read length of hybridization sequencing is defined by the length of the oligonucleotide probe. In hybridization sequencing, the sample DNA first has to be prepared, extracted and also amplified using PCR before sequencing can be carried out.
Currently, two companies, Affymetrix and Perlegen, have developed platform for use with hybridization sequencing. The platform functions by first hybridizing the DNA sample to microfabricated arrays of immobilized oligonucleotide probes which contain four different features. Each feature contains a different nucleotide, adenine, cytosine, guanine, and thymie. The base pair will bind to the complementary feature. Each of the four features will then exhibit a signal and the strongest signal determines which of the four features the base pair has bound on to. To enable sequencing of large organisms, the use of DNA ligase together with hybridization sequencing was developed. This enabled hybridization sequencing to perform rapid and large-scale DNA sequencing. By using this method, DNA sample can be rapidly sequenced. Other platforms which are available in the market are Agilent, Applied Biosystems, and Compuged, although Affymetrix remains the most widely used platform up to date.
Current studies in the field of hybridization sequencing are focused on creating a probe or a strategy which would ensure that cross-hybridization of probes due to repetitive elements would not occur during sequencing. Previous studies using SNP showed a 3% false positive rate due to repetitive elements. Another challenge was ensuring that the sample DNA binds only to one probe. This can be overcome by attaching dendritic molecules to the surface of the probes as this will increase the distance between each probe. If these challenge can be overcome, hybridization sequencing could have a huge potential for sequencing of large organisms, for example humans as hybridization sequencing is rapid and cost effective as reagent are not needed due to the use of microfabricated arrays.
With the development of advanced sequencing technologies, is it hoped that genomes would be able to be sequenced in individual laboratories instead of involving large production scale effort as the cost of sequencing large amounts of genomes are being reduced. This would prove to be a milestone in accelerating biological, biotechnological, and biomedical research. Advancement in DNA sequencing technologies are essential in order to enable accurate and efficient sequencing of large scale and entire genomic material. Although genome sequencing had advanced in leaps and bounds since Sanger sequencing, progress can still be made, with the use of more advanced material such as nanotubes and nanomaterials. Also, focus should shift from creating new technologies to improving and mastering existing technologies in order to harness the full potential which each technology has to offer in terms of DNA sequencing. This would enable scientist to gain valuable information from large amounts DNA.