NANOPORE SEQUENCING GROUP ASSIGNMENT

NANOPORE SEQUENCING Basic components of nanopore

CONTENTS
1. Background
2. Components
3. Mechanism
4. Advantages
5. Applications
6. Limitations
7. Conclusion
Background
•FOUNDATION
• David Deamer -1990. Drove a damaged RNA
through a protein pore to synthesize it from scratch
• Hagan Bayley- 1990.Studied transmembrane
protein alpha hemolysin & Developed a stochastic
sensor
• George Church – 1995. HGP. An embedded
electronic monitored device on a membrane can
provide a sequence information)
• Collaborations lead to the modern nanopore
sequencer - 2004 • Figure 1:WIKIPEDIA, D.Ds notebook
Background
•What is nanopore sequencing ?
• A technique of analysing nucleotides.
• A Nucleotide is driven through the pore,
causing a disturbance on the ionic solution- the
disturbance signals the nucleotide sequence
• Two types
• Biological
• Solid state
Background- nanopore
sequencing types

Figure 2: wikipedia, nanopore sequencing types

 Biological nanopore
• transmembrane protein channel
• Natural
• Limited size 1.4nm

BACKGROU
ND • Solid state
• Silicon compound (Si3N4)channel
• Synthesised
• Must be > 2nm
BACKGROUND

• Additional comparison types

COMPONENTS
• Analyte- Negatively charged DNA or RNA strand and oligonucleotide proteins.
• Nanopore- A biological or solid state (silicon compound) forming a gateway for
the analyte.
Diameter 2.6nm, Length 10nm
• Membrane (lipid bilayer) – Embeds the nanopore.
High electric resistance.
Immersed in an electrophysiological solution
• Motor enzyme (protein)- 1 to 450 nucleotide per second
• Adapter- Hairpin like structure,
Assistance in DNA movement and feeding into the nanopore correctly.
MECHANISM
• Motor protein.
Assistance in DNA movement into membrane,
Helps translocase to capture the molecule being analysed.
DNA speed, 450 bp/sec. RNA speed, 70 bp/sec
Provided on an adapter up to the end of the DNA or RNA strand.
• The helicase enzyme unzips the double stranded DNA.
• The translocase enzyme assists in the movement of the DNA.
• Adapter library
• DNA single strand fed through the nanopore sequencing the intact strand.
• Applied current causes the DNA to move through the pore to the other side of the chamber.
• A flow of ions through the pore creates a current
MECHANISM
• Current is disrupted depending on the base pair(characteristically).
• Current modulator takes the reading.
• The magnitude of the current depends on the base composition, shape,
size and length of the DNA.
• A signal is created and compared to known nucleotide measurements.
• From this an interpretation is made, thus getting the identity of the
analyte.
• The nanopore becomes available for another nucleotide as soon as one
the one that was inside the pore is completely out and analysed. Single
nucleotide at a time
 Advantages
• Nano-pore devices are relatively small, and can be powered by a USB port
• Previously necessary in-laboratory sequencing is no longer necessary
• Advancing features resulted in increased base throughput (base reading
rate) and accuracy even for complex genomes
• Advanced nano-pore devices can sequences, sequences greater than 100
kilo-bases
• Sequencing costs start from as little as $ 1000.00
 Application
Nano-pore sequencing has opened several research opportunities in Biological
Science
Most noticeable applications:
• The real time-sequencing in sequencing in severe outbreaks of Ebola virus
• The identification and analysis of antibiotic resistant profiles in bacteria
• Detection of pathogens in blood samples
• Pre-natal screening or testing
• A quarter of the worlds SARS-Cov 2 virus genome has been sequenced using it
DISADVANTAGES & LIMITATIONS
• Nanopore sequencing tends to be prone to error
• Error rate up to 15%
• Can be tolerable
• E.g. self complementary binding
• Biological nanopores have environmental demands and these keep
the biological activities running
• Temperature, concentration and pH
• They have low read accuracy as compared to short read sequencers
• Single nucleotide variation
Conclusion
• Would you advice someone to use nanopore sequencing?
• What needs to be done to improve it?
• What does improving nanopore sequencing depend on?
 Branton, D., Deamer, D.W., Marziali, A., Bayley,
H., Benner, S.A., Butler, T., Di Ventra, M.,
Garaj, S., Hibbs, A., Huang, X. and Jovanovich,
S.B., 2008. The potential and challenges of
nanopore sequencing. Nature
biotechnology, 26(10), pp.1146-1153.
REFERENCE
S A. M. Zaki, S. van Boheemen, T. M. Bestebroer,
A. D. M. E. Osterhaus, and R. A. M. Fouchier,
“Isolation of a novel coronavirus from a man
with pneumonia in Saudi Arabia,” New
England Journal of Medicine, vol. 367, no. 19,
pp. 1814–1820, 2012.