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Second generation DNA-Sequencing Technique-HeliScope Bioscience - Coggle…
Second generation DNA-Sequencing Technique-HeliScope Bioscience
References:
Athanasopoulou, K., Boti, M. A., Adamopoulos, P. G., Skourou, P. C., & Scorilas, A. (2021). Third-Generation Sequencing: The Spearhead towards the Radical Transformation of Modern Genomics. Life, 12(1), 30.
https://doi.org/10.3390/life12010030
Crosetto, N., Mitra, A., Silva, M. J., Bienko, M., Dojer, N., Wang, Q., Karaca, E., Chiarle, R., Skrzypczak, M., Ginalski, K., Pasero, P., Rowicka, M., & Dikic, I. (2013). Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nature Methods, 10(4), 361–365.
https://doi.org/10.1038/nmeth.2408
Hart, C., Lipson, D., Ozsolak, F., Raz, T., Steinmann, K., Thompson, J., & Milos, P. M. (2010). Single-Molecule Sequencing. Methods in Enzymology, 407–430.
https://doi.org/10.1016/s0076-6879(10)72002-4
Kchouk, M., Gibrat, J. F., & Elloumi, M. (2017a). Generations of Sequencing Technologies: From First to Next Generation. Biology and Medicine, 09(03).
https://doi.org/10.4172/0974-8369.1000395
Kchouk, M., Gibrat, J. F., & Elloumi, M. (2017b). Generations of Sequencing Technologies: From First to Next Generation. Biology and Medicine, 09(03).
https://doi.org/10.4172/0974-8369.1000395
Milos, P. M. (2010). Helicos single molecule sequencing: unique capabilities and importance for molecular diagnostics. Genome Biology, 11(Suppl 1), I14.
https://doi.org/10.1186/gb-2010-11-s1-i14
Pareek, C. S., Smoczynski, R., & Tretyn, A. (2011). Sequencing technologies and genome sequencing. Journal of Applied Genetics, 52(4), 413–435.
https://doi.org/10.1007/s13353-011-0057-x
Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature Biotechnology, 26(10), 1135–1145.
https://doi.org/10.1038/nbt1486
Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next-generation sequencing technologies for environmental DNA research. Molecular Ecology, 21(8), 1794–1805.
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Thompson, J. F., & Steinmann, K. E. (2010). Single Molecule Sequencing with a HeliScope Genetic Analysis System. Current Protocols in Molecular Biology, 92(1).
https://doi.org/10.1002/0471142727.mb0710s92
Advantages and Disadvantages
Disadvantages
• The yield of sequences per unit mass is dependent on the number of 3′ end hydroxyl groups and thus having relatively short templates for sequencing is more efficient than having long templates.
• The error rate is high due to noise. This can be overcome with repetitive sequencing but increases the cost per base for a given accuracy rate, offsetting some of the gains from lower reagent costs. The raw read error rates are generally at 5%, although the highly parallel nature of this technology can deliver high fold coverage and a consensus or finished read accuracy of 99%.
Advantages
• Sample preparation does not require ligation or PCR amplification, avoids PCR-induced bias and errors, simplifies data analysis and tolerates degraded samples. Instead, it sequences the DNA strand itself, enabling direct, high-precision measurement.
• Is capable of sequencing nucleic acids over a very broad range of template lengths, from several nucleotides to several thousand nucleotides without the need for size selection in most situations.
• Can directly read the sequence of a single such strand, a capability that gives it the potential for unprecedented speed. Single-molecule sequencing may be able to generate a more complete picture of the genome. That’s because when DNA is amplified, some strings are likelier than others to be copied successfully, so they’re more likely to be represented in the final sequence.
Background
As what stated on the research paper published in Proceedings of the National Academy of Sciences (PNAS), it is the first commercial machine to provide sequencing on single strand DNA.
Does not require high cost; able to provide a faster and cheaper sequencing technique.
Consists of an instrument called Helicos sequencer that able to carry out sequencing on a single strand DNA which uses synthesis technique.
Helicos Bioscience Corporation developed nonstick material in contrasting the unamplified fluorescent signal of single base in growing DNA strand.
Able to resequence each individual template in situ using a single-molecule technique, which significantly lowered the ensemble error rate.
This sequencing method and equipment are used to sequence the genome of the M13 bacteriophage.
Different between Sanger sequencing, Second generation sequencing and Third generation sequencing
Sanger Sequencing
. Known as "chain termination method" to determine the nucleotide sequence of DNA.
. Need to performe it manually or usually in an automated fashion vie sequencing machine
. Works same as standard PCR but with one difference which is the addition of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs)
. Need to use gel electrophoresis for analyzing the data
. Low throughput and high cost of these time-consuming method
Third Generation Sequencing
. Novel set of sequencing, Pacific Bioscience(PacBio) and Oxford Nanopore Sequencing(ONT)
. Better than the second generation sequencing because of two features which are single-molecule sequencing (SMS) and sequencing in real-time
. Allow the sequencing of nucleotide molecules (DNA or RNA) without the need for PCR amplification of the template
. The new developed sequencing protocols by Third generation sequencing make the sequencing method easier because of simplified library construction processes, minimizing their preparation time and making sequencing runs an ease-of-handle and also time-saving procedure
. The ONT also developed the pocket-sized sequencer that enable the user to perform a sequencing run anywhere and right after done the environmental sample collection
Second Generation Sequencing
. Most used method in the world
. The generation of many millions of short reads in parallel
. Low cost of sequencing and less time consuming compared to the first generation
. The sequencing output is directly detected without the need for electrophoresis
. Roche/454 sequencing, Illumina/Solexa, ABI/SOLiD and Ion torrent sequencing is the company that developed the machine for this generation
. Less error, most of it just has 1% error chance
The mechanism of 2nd Generation DNA Sequencing Technique (Heliscope Bioscience)
Library fragmentation
Random fragmentation and poly-A are captured by hybridization to surface-tethered poly-T oligomers to yield a disordered array of primed single-molecule sequencing template (Shendure & Ji, 2008).
Hybridization of DNA fragments to the poly-T oligonucleotides which are attached to the flow cell and simultaneously sequenced in parallel reactions (Pareek et al., 2011).
Sequencing cycle
Repetitive DNA polymerase cycles and one of four fluorescently labelled nucleotides are flowed in (Shokralla et al., 2012).
Fluorescent nucleotides are modified to stop the polymerase extension until the incorporated nucleotide's fluorescence is captured and images are recorded with a highly CCD camera connected to fluorescence microscope (Shokralla et al., 2012).
Sequencing can be up to 28 Gb in a single sequencing run and takes about 8 days. Generate short reads of maximal length 55 bases.
New generation of
one-base-at-a-time
allows more accurate homopolymer and direct RNA sequencing.
Detection of incorporated nucleotides
Nucleotide detection via HeliScope sequencer.
Subsequent chemical cleavage of fluorophores allows next cycle of DNA elongation to begin with another fluorescently labelled nucleotide (Pareek et al., 2011).
Enable determination of DNA sequence.
Principles
Unrequired amplification step.
True single molecule sequencing (tSMS) technology.
How to analyse data
Individual strands of DNA will be collected and then will be deposited onto the Helicos Flow Cell glass surface that coated with oligo-dT-50 oligonucleotides and filled with dTTP and polymerase.
The samples were then loaded into the Helicos Sample Loader and the temperature can be adjusted for optimal hybridization.
After the flow cells have been properly filled with sample, they are placed in the HeliScope Sequencing System with with all of the reagents required for synthesis and image sequencing.
The HeliScope Analysis Engine will process the image of sequencing in real time and we can read the sequence reads that were built during the process.
The completed data will be downloaded into the computer cluster for further action; either for reference alignment or for assemble.