Nanopore Technology is the fourth generation polynucleotide sequencing technique. Note: For educational purpose only.

1. NANOPORE SEQUENCING
Somesh Shingane
Kuldeep Sharma
M.Tech Biological Engineering, IIT GANDHINAGAR.

2. Introduction
Fourth Gen Sequencing Polynucleotide Sequencing Single Molecule Detection
Constantly Evolving Nanometer Scale Pore Size

3. Background and Principle behind
Nanopore Sequencing
 Around 1980s David Deamer had an implausible suggestion that it might be possible to
sequence a single strand of DNA(ssDNA) by being drawn through a membrane’s
nanoscopic pore by the process of electrophoresis.
 George Church’s interest in scaling up DNA sequencing for the HGP led him to propose
that an electronically monitored embedded system across bilayer membrane might
provide sequence information upon processing of DNA.
 Hagan Bayley’s interest in membrane proteins and the structure and assembly of
oligomeric transmembrane protein pores, such as α-hemolysin, also led him and his
colleagues to ask if pores could serve as biosensors of molecules and cation.
 With the collaboration of David Deamer ,John Kasianowicz together with Sergey
Bezrukov at the US National Institutes of Health (NIH) studied the structure of α-
hemolysin and compared with size of dsDNA(it was perfect fit!)

4. Pages from the book of David Deamer

5. Basics of Nanopore Sequencing

6. But…
 How do we know only ssDNA can pass through the nanaopore?
 How can we make sure that ssDNA is guided to the pore without any hindrances?
 How do we differentiate between the purines and pyrimidines just by the current signals when
polynucleotide passes through the pores?
 How much time does it takes to identify the sequence and how many base pairs can it identify at a
time or how much it should be?
 Is α-hemolysin the only nanopore for the sequencing purpose?

7. Timeline of Nanopore Sequencing

8. Minds Behind the Idea of Nanopore Sequencing

9. Construction
Sensor Cell
Electric
reader unit
Chambers
separated by
septum; Lipid
bilayer; electrode
o Transmembrane Protein
subunit
o Nucleic acid handling
enzyme
o Membrane unit

10. Transmembrane Protein
Other Proteins : OmpF, OmpG, OmpA, NaLP, WZA

11. NA Handling Enzyme
Phi29 DNA
Polymerases RecD Helicases
Source: PDB

12. Membrane types
Biological
Solid State

14. Oxford Nanopore Technologies

15. Advancement
 Covalent linkage of transmembrane subunit to NA handling enzyme (Patent Application No. WO2010004265A1)
 SSB use to prevent secondary structure formation (Patent Application No. WO2014013259A1)
 Coupling the target polynucleotide to the membrane (Patent Application No. WO2012164270A1)
 Hairpin loop method for dsDNA sequencing (Patent Application No. WO2013014451A1)

16. Pros and Cons Nanopore Sequencing
Pros Cons
• Minimum amount of data processing and complex
algorithm are required.
• The DNA sequence analyzing system is yet to reach
its maximum efficiency.
• Can identify the genomic sequence at large scale (
upto gigabase pairs) at one go.
• Handling the long strand of ssDNA is also a major
concern i.e. to avoid self complimentary binding.
• The size of pore is fixed and does not vary for large
variation of environment or eperimental.
• Though it can work in various experimental
conditions yet it has some temperature limitations.
• Real time sequencing at high rate of accuracy (upto
98%)
• Controlling the speed of ssDNA passing through the
pore is yet to be achieved at maximum efficiency.
• Can sequence the entire human genome in less than
$1000
• High signal to noise ratio is yet to obtain.
• Translocation of polynucleotide through pores are in
precise and control manner.
• Number of run per pore membrane is also bit low.

17. THANK
YOU