Nanopore DNA Sequencing (NGS)

1. NANOPORE DNA SEQUENCING
1
Submitted By:-
Sourabh kumar
Department of Genetics & Plant Breeding(CCSU.Meerut.)
Seminar
on

2. OUTLINE
Introduction & Milestones
 Application & Types
Different methods
-Using α-Hemolysin
-Using MspA
-Using Graphene etc……
Advantage & Disadvantage
Challenges & Conclusion
References
2

3. DNA SEQUENCING
• DNA sequencing is the
process of determining
the precise order of
Nucleotides within a DNA
molecule. may be genes
or entire genomes.
• Four categories:-
1.SBH, 2.SBS, 3.SBL,
4.Nanopore Sequencing.
3

4. Milestones in nanopore DNA sequencing:-
NATURE BIOTECHNOLOGY VOLUME 34 NUMBER 5 MAY 2016,
Page No.519
4

5. WHAT IS A NANOPORE?
Nanopore = ‘very small hole’
Electrical current flows through the hole
Introduce analyte of interest into the hole 
identify “analyte” by the disruption or block to
the electrical current
Current
flow
5

6. Principles for detection and base identification:-
Nanopore sequencing uses electrophoresis to transport an unknown sample. A nanopore system
always contains an electrolytic solutions- when a constant electric field is applied, an electric
current can be observed in the system. The magnitude of the electric current density across a
nanopore surface depends on the nanopore's dimensions and the composition of DNA or
RNA that is occupying the nanopore. Sequencing is made possible because, when close enough to
nanopores, samples cause characteristic changes in electric current density across nanopore surfaces.
The total charge flowing through a nanopore channel is equal to the surface integral of electric current
density flux across the nanopore unit normal surfaces between times t1 and t2.
When the nanopore is of molecular dimensions, passage of molecules (e.g., DNA) cause interruptions
of the current level, leading to a signal.

7. APPLICATION OF NANOPORE
Sensor array chip: many nanopores in parallel
DNA Sequencing Proteins Polymers Small Molecules
Adaptable protein nanopore:
Application
Specific
Generic
Platform
Electronic read-out system
7

8. In Nanopore DNA Sequencing:-
• Nanopore sequencing is a fourth generation approach used
in the sequencing of biopolymers-
specifically, polynucleotides in the form of DNA or RNA.
• Using nanopore sequencing, a single molecule of DNA or
RNA can be sequenced without the need
for PCR amplification or chemical labeling of the sample.
• Nanopore sequencing has a very simple basic
principle, DNA strands or single nucleotides are
driven through a nanopore electrophoretically.

9. In Nanopore DNA Sequencing:-
• Since DNA bases (adenine, thymine, cytosine and
guanine) are different from each other in atomic
scale, it is essential to collect base-specific
information at atomic level to correspond the
DNA sequence with the measured signals.
Obtained to the different types of the signals,
which is detected by electrical detection method.

13. Nanopore technologies can be broadly divided
into two categories:
1. Biological nanopore
2. Solid-state nanopore
More recently, hybrid nanopores have been
proposed to take advantage of the features
of both biological and solid-state nanopores.
TYPES OF NANOPORES

14. 1.ALPHA-HEMOLYSIN
• a-HL is an exotoxin secreted by
the bacterium Staphylococcus
aureus, a human pathogen.
• This mushroom-shaped
heptameric protein normally
forms pores in the membranes.
• The limited pore size (1.4 nm)
Biological nanopores
 also called Transmembrane protein channels,
 are usually inserted into a substrate, such as planar lipid bilayers, liposomes,
or other polymer films.
Nanopores formed by pore-forming proteins:-
Examples:

15. Mycobacterium smegmatis porin A
(MspA)
• Powerful nanopore for
reading information from
four nucleotides
simultaneously.
• by Butler et al. in 2008.
• The channel of the MspA
octamer is 1 nm in diameter
at the minimal point, which
is relatively small and
narrow, compared to that of
alpha-HL. Thus, it can
improve the spatial
resolution of ssDNA
sequencing.

16. 2.SOLID STATE NANOPORE
• In 2001, Li et al.
• are made in
silicon compound
membranes
(Silicon nitride)
• More recently, the
use of graphene
as a material for
solid-state
nanopore sensing
has been
explored.

17. The portable MinION –– the first
handheld nanopore DNA sequencer

20. The significant
advantages of nanopore seq.
• Label-free,
• Ultra-long
• High throughput, and
• Low material requirement.
• The nanopore approach is one option for the
fourth-generation low-cost and rapid DNA
sequencing technology.

21. Challenges:
• To slow down DNA translocation from
microseconds per base to milli seconds.
• To reduce stochastic motion of the DNA molecule
in transit in order to decrease the signal/noise
ratio

22. Role of NGS in genomics-assisted breeding.
Varshney RK,Terauchi R, McCouch SR (2014) Harvesting the Promising Fruits of Genomics: Applying Genome Sequencing
Technologies to Crop Breeding. PLOS Biology 12(6): e1001883. https://doi.org/10.1371/journal.pbio.1001883
http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1001883

23. References
• Deamer, D., Akeson, M. and Branton, D., 2016.
Three decades of nanopore
sequencing. Nature biotechnology, 34(5),
pp.518-524.
• Plant Biotechnology-Prop. P.K.Gupta
• Prof.P.K.Gupta,Trends in Biotechnology Vol.26
No.11
• Internet

24. Thanks