Nanopore sequencing (NGS)

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 current in out 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 t 1  and t 2 . 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.

10. Nanopore technologies can be broadly divided into two categories: Biological nanopore 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

11. 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:

12. 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.

13. 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.

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

15. 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 .

16. 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

17. 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

18. 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

19. Thanks