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Third generation DNA sequencing technologies (Oxford Nanopore (Advantages,…
Third generation DNA sequencing technologies
Oxford Nanopore
Generations of Sequencing Technologies: From First to Next Generation
https://www.omicsonline.org/open-access/generations-of-sequencing-technologies-from-first-to-next-generation-0974-8369-1000395.php?aid=87862
(March 06, 2017)
The Oxford Nanopore sequencing (ONT) was developed as a technique to determine the order of nucleotides in a DNA sequence.
(2015) Genome assembly using Nanopore-guided long and error-free DNA reads.(
https://www.ncbi.nlm.nih.gov/pubmed/25927464
)
Advantages
No library prep required
Long reads lengths
Protein-->solid-state upgrades may eliminate reagent costs
Fast turn around
Could measure epigenetic modifications and other molecules
Disadvantages
Potentially non-stochastic errors
Difficult to see how the same molecule could be sequenced repeatedly
Platform
Minlon for individual
(2016)Oxford Nanopore MinION Sequencing and Genome Assembly. (
https://www.ncbi.nlm.nih.gov/pubmed/27646134
)
GridION for sequencing centres
(2017) A world of opportunities with nano pore sequencing.(
https://academic.oup.com/jxb/article-abstract/68/20/5419/4093050?redirectedFrom=fulltext
)
Oxford Nanopore passes an ionic current through nanopores and measures the changes in current as biological molecules pass through the nanopore or near it.
PacBio RS
Advantage
Longer reads lengths(200bp-10kb)
40 minutes run time
Same molecular can be sequenced repeatedly
Epigenetic modifications can be detected
K.Nakano, A.Shiroma(2017)Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area (
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486853/
)
produce genome sequence data containing fewer gaps and longer contigs
Pacific Bioscience SMRT
A.McCarthy (2010) Third Generation DNA Sequencing:
Pacific Biosciences’ Single Molecule Real Time Technology. (
https://www.sciencedirect.com/science/article/pii/S1074552110002474?via%3Dihub
)
Disadvantage
Library prep required
Enzyme based
Only 20k-75k reads per run initially
High error rate per run
expensive
Application
R.Anthony, A.F.Kin (2015) PacBio Sequencing and Its Applications(
https://www.sciencedirect.com/science/article/pii/S1672022915001345
)
utilized to study much larger genomes including human
De novo genome assembly, because of long reads can provide large scaffolds. and long read can overcome many limitations of genome assembly
characterization of structural variation,
Platform
M.Ferrarini, M.Moretto (2013)An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome.(
https://www.ncbi.nlm.nih.gov/pubmed/24083400
)
1) increase in read length from tens of bases to tens of thousands of bases per read; (2) reduction of sequencing time from days to hours (or to minutes for real-time applications); and (3) reduction or elimination of sequencing biases introduced by PCR amplification
single molecule sequencing