Single molecule sequencing foremost made its introduction in 2003 and is based on the rule of asynchronous synthesis. Aside from sequencing genomes, asynchronism could besides be used to help with the reading of base repetition sequences and homopolymers. Single molecule sequencing capitalizes on the fact that this engineering does non necessitate genomic Deoxyribonucleic acid to be cloned, amplified, or ligated before it is sequenced. This will enable the cost and clip required for genomic sequencing to be made more low-cost, while supplying high throughput sequencing engineering.
Surveies conducted by Pushkarev et al. , 2009, demonstrated that individual molecule sequencing was really accurate. Pushkarev et al. , 2009 sequenced a complete human genome, about 3.8million base brace, utilizing individual molecule sequencing and found that the consequences obtained had a false-positive rate of less than 1 % . The low rate of errors was possible as each templet molecule was examined individually from other templet molecules. This provided the advantage of low rate of mis-incorporation of the incorrect base as the dynamicss of base incorporation do non vie with the dynamicss of templet molecule reading. Single molecule sequencing besides allows templet molecules to be sequenced in situ, which would besides cut down the rate of misincorporation of bases.
Helicos Bioscience was the first research company to let go of a platform, Heliscope Single Molecule Sequencing, which enabled genome to be sequence utilizing sequencing-by-synthesis processs. Heliscope Single Molecule Sequencing enables up to 1 billion base braces to be sequenced within a hebdomad. This would enable rapid and big graduated table sequencing of genomes, including sequencing of the human genome, which was demonstated by Pushkarev et al. , 2009. Heliscope Single Molecule Sequencing operates by sequencing single nucleic acid molecules in DNA or RNA which were foremost melted into individual stranded Deoxyribonucleic acid and poly-A tail. The individual stranded Deoxyribonucleic acid are so placed onto the surface of the glass Heliscope Flow Cell which was coated with oligonucleotide. The Heliscope Flow Cell is so filled with dTTP and polymerase in order to make full any staying bases which are complementary to the poly A-tail. Then, fluorescently labeled base is added and sequencing is carried out. Fluorescently labeled base which is complementary to the templet DNA is detected by emanation of fluorescent visible radiation one time optical maser is shown through the flow-cell surface. Heliscope so captures the image and enter the place of the fluorescent bases, following which the fluorescence will be cleaved from the integrated bases. The rhythm is so repeated, with Heliscope Single Molecule Sequencing continually adding in complementary bases in following tally rhythms.
Although individual molecule sequencing has many advantages, individual molecule sequencing does hold its ain reverses. It was found that when SNPs were being sequenced, presence of nonzero false-positive and false-negative rates were high.
Another advanced sequencing engineering which has gained popularity is existent clip individual molecule DNA sequencing. DNA polymerase was chosen as the method used for existent clip individual molecule sequencing as DNA polymerase is a stable, single-subunit enzyme that has a rapid, efficient, high processivity rate for doubling genomes. As Deoxyribonucleic acid polymerases have proofreading activity, the mistake rates exhibited were really low, about one in every 105 bases.
An advantage of utilizing DNA polymerase was that DNA polymerase would be able to sequence near round templets multiple times in a individual tally. This would cut down the cost of sequencing the genome as merely one DNA molecule was needed to find a round consensus sequence. Reagent ingestion would besides be minimized as merely little sums of genomic DNA are required for a individual tally. Real clip individual molecule sequencing would besides diminish the sum of clip needed to sequence a genome by about four orders of magnitude compared to Sanger sequencing. This was because the existent clip individual molecule sequencing enables 1000s of bases to be read at the same time due to uninterrupted DNA synthesis.
The platform used for existent clip individual molecule existent clip sequencing was developed by Pacific Biosciences. The instrument is a nanophotonic construction which uses high-multiplex confocal fluorescence sensing system that targets unvarying multilaser light of 3000 zero-mode wave guide ( ZMWs ) through holographic stage masks. This instrument uses a confocal pinhole array to reject out-of-focus background, and a prism diffusing component for wavelength favoritism. This would enable rapid and accurate sequencing while supplying flexibleness in the pick of fluorescent dyes used for conveying up to 99 % of the incident visible radiation.
It was found that if genome was sequenced utilizing existent clip individual molecule sequencing, mistakes were detected due to omissions which occurred due to unlabelled nucleotide taint. This mistake could be overcomed by utilizing a dNTP composing which was more than 99.5 % pure. Another method would be to cut down the fraction of short incorporations events in the genome, addition fluorophore brightness and better efficiency of light aggregation of the sensing system.
Surveies conducted by Eid et al. , 2009, showed a 99.3 % truth of the genome sequenced. In order to increase the truth of the genome being sequenced, different dyes and soaking up wavelengths could be used. Dyes which provide larger spectral separations while at the same clip increasing the brightness of the instrument would supply higher truth of the genome being sequenced. The merged of rapid individual molecule sequencing and existent clip DNA sequencing enabled rapid, big graduated table and cost-efficient sequencing of genomes. This would let independent research labs to transport out research which was antecedently merely carried out by major genome institutes.
Hybridization sequencing sequences DNA based on the rule that different oligonucleotides will crossbreed on different investigations. The advantage of utilizing hybridisation sequencing is that a big sum of sequences could be obtained from the genome in a comparatively short sum of clip. This was because hybridisation sequencing utilizations microarrays, which are able to read up to 109 base brace. The read length of hybridisation sequencing is defined by the length of the oligonucleotide investigation. In hybridisation sequencing, the sample DNA foremost has to be prepared, extracted and besides amplified utilizing PCR before sequencing can be carried out.
Presently, two companies, Affymetrix and Perlegen, have developed platform for usage with hybridisation sequencing. The platform maps by first crossbreeding the Deoxyribonucleic acid sample to microfabricated arrays of immobilized oligonucleotide investigations which contain four different characteristics. Each characteristic contains a different base, A, C, G, and thymie. The base brace will adhere to the complementary characteristic. Each of the four characteristics will so exhibit a signal and the strongest signal determines which of the four features the base brace has bound on to. To enable sequencing of big beings, the usage of DNA ligase together with hybridisation sequencing was developed. This enabled hybridisation sequencing to execute rapid and large-scale DNA sequencing. By utilizing this method, DNA sample can be quickly 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 day of the month.
Current surveies in the field of hybridisation sequencing are focused on making a investigation or a scheme which would guarantee that cross-hybridization of investigations due to insistent elements would non happen during sequencing. Previous surveies utilizing SNP showed a 3 % false positive rate due to insistent elements. Another challenge was guaranting that the sample DNA binds merely to one investigation. This can be overcome by attaching dendritic molecules to the surface of the investigations as this will increase the distance between each investigation. If these challenge can be overcome, hybridisation sequencing could hold a immense potency for sequencing of big beings, for illustration worlds as hybridisation sequencing is rapid and cost effectual as reagent are non needed due to the usage of microfabricated arrays.
With the development of advanced sequencing engineerings, is it hoped that genomes would be able to be sequenced in single research labs alternatively of affecting big production graduated table attempt as the cost of sequencing big sums of genomes are being reduced. This would turn out to be a milepost in speed uping biological, biotechnological, and biomedical research. Advancement in DNA sequencing engineerings are indispensable in order to enable accurate and efficient sequencing of big graduated table and full genomic stuff. Although genome sequencing had advanced in springs and bounds since Sanger sequencing, advancement can still be made, with the usage of more advanced stuff such as nanotubes and nanomaterials. Besides, focal point should switch from making new engineerings to bettering and get the hanging bing engineerings in order to tackle the full potency which each engineering has to offer in footings of DNA sequencing. This would enable scientist to derive valuable information from big sums Deoxyribonucleic acid.
