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Oxford Nanopore
Sequencing
Presented by,
P. Sangeetha
M.Sc. Microbiology
19308021
Introduction
• In 2005, Oxford Nanopore was founded in
Oxford Nanolabs by Dr.Gordon
Sanghera, Dr.Spike
Willcocks and Professor Hagan Bayley
(currently Professor of Chemical Biology at
the University of Oxford),
• Nanopore sequencing has been around
since the 1990s, when Church et al. and
Deamer and Akeson separately proposed
that it is possible to sequence DNA using
nanopore sensors.
Nanopore sequencing
• Each base produces different ionic current
torrents during DNA translocating through a small
channel (nanopore), and these changes can be
used to determine the sequence of the nucleic
acid.
• The pores are usually in a biological membrane,
or in solid state film.
• The hole is blocked, due to passage of molecules.
The current flow is also blocked. Each nucleotide
blocks the channel differently giving a different
amplitude and duration of the blockade. This
information is converted into DNA sequence
information.
Nanopore DNA sequencing
• Strand sequencing:
– Sequencing in real time
as the intact DNA
polymer passess
through the nanopore.
• Exonuclease
sequencing:
– Individual nucleotides
pass through the
nanopore by the aid of
processive
exonuclease.
Oxford Nanopore
Sequencing
Oxford Nanopore
sequencing
ss DNA or
RNA
Exonuclea
se- PNAs
Nanopore
Adapter
Detection
Polynucleotide phosphorylase
(PNPs)
 PNPs cleaves ssRNA in the 3’ to 5’
direction using inorganic phosphate to
attack the phosphoester linkage and
liberate rNDPs.
Oxford Nanopore
• Alpha-Hemolysin (obtained from)
• The nanopore through the which the ssDNA or RNA is
sequenced though is a alpha hemolysin protein mutant
(M113R-RL2 ).
• Mutants of the α-hemolysin (αHL) pore that bind the
adapter β-cyclodextrin (βCD) ∼104 times more tightly than
the wild type have been obtained.
• Mutant pore, M113N7, that
releases βCD ∼104 times
more slowly
Adapters
– Adapter provides internal binding site for small
molecules
– am7βCD adapter (heptakis-(6-deoxy-6-amino)-β-
cyclodextrin; which comprise seven primary hydroxyls
replaced with amino groups.
– gu7βCD adapter (heptakis(6-deoxy-6-guanidino)-
βCyclodextrin; The molecule comprises βCD with seven
guanidinium groups in place of
the primary hydroxyls, with the
guanidinium groups
Detection of rNDPs/dNDPs:
Advantages
• Potential, quickly, reliable
• The entire Human genome sequenced for
less than $750
• Read length 50-100 kbs.
• The first product, MinION, was
introduced into early access in 2014
and made commercially available in
2015.
• Portable device.
Reference:
• Nanopore-based identification of individual
nucleotides for direct RNA sequencing Mariam
Ayub, Steven W. Hardwick, Ben F. Luisi, and Hagan
Bayley.
• Ayub, M. and Bayley, H. (2012). Single Molecule
RNA Base Identification with a Biological
Nanopore.Biophysical Journal 102:429.Oxford
Nanopore.
Thank you

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Oxford nanopore sequencing

  • 1. Oxford Nanopore Sequencing Presented by, P. Sangeetha M.Sc. Microbiology 19308021
  • 2. Introduction • In 2005, Oxford Nanopore was founded in Oxford Nanolabs by Dr.Gordon Sanghera, Dr.Spike Willcocks and Professor Hagan Bayley (currently Professor of Chemical Biology at the University of Oxford), • Nanopore sequencing has been around since the 1990s, when Church et al. and Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors.
  • 3. Nanopore sequencing • Each base produces different ionic current torrents during DNA translocating through a small channel (nanopore), and these changes can be used to determine the sequence of the nucleic acid. • The pores are usually in a biological membrane, or in solid state film. • The hole is blocked, due to passage of molecules. The current flow is also blocked. Each nucleotide blocks the channel differently giving a different amplitude and duration of the blockade. This information is converted into DNA sequence information.
  • 4. Nanopore DNA sequencing • Strand sequencing: – Sequencing in real time as the intact DNA polymer passess through the nanopore. • Exonuclease sequencing: – Individual nucleotides pass through the nanopore by the aid of processive exonuclease.
  • 6. Oxford Nanopore sequencing ss DNA or RNA Exonuclea se- PNAs Nanopore Adapter Detection
  • 7. Polynucleotide phosphorylase (PNPs)  PNPs cleaves ssRNA in the 3’ to 5’ direction using inorganic phosphate to attack the phosphoester linkage and liberate rNDPs.
  • 8. Oxford Nanopore • Alpha-Hemolysin (obtained from) • The nanopore through the which the ssDNA or RNA is sequenced though is a alpha hemolysin protein mutant (M113R-RL2 ). • Mutants of the α-hemolysin (αHL) pore that bind the adapter β-cyclodextrin (βCD) ∼104 times more tightly than the wild type have been obtained. • Mutant pore, M113N7, that releases βCD ∼104 times more slowly
  • 9. Adapters – Adapter provides internal binding site for small molecules – am7βCD adapter (heptakis-(6-deoxy-6-amino)-β- cyclodextrin; which comprise seven primary hydroxyls replaced with amino groups. – gu7βCD adapter (heptakis(6-deoxy-6-guanidino)- βCyclodextrin; The molecule comprises βCD with seven guanidinium groups in place of the primary hydroxyls, with the guanidinium groups
  • 11. Advantages • Potential, quickly, reliable • The entire Human genome sequenced for less than $750 • Read length 50-100 kbs.
  • 12. • The first product, MinION, was introduced into early access in 2014 and made commercially available in 2015. • Portable device.
  • 13. Reference: • Nanopore-based identification of individual nucleotides for direct RNA sequencing Mariam Ayub, Steven W. Hardwick, Ben F. Luisi, and Hagan Bayley. • Ayub, M. and Bayley, H. (2012). Single Molecule RNA Base Identification with a Biological Nanopore.Biophysical Journal 102:429.Oxford Nanopore.