Nanopore sequencing

Nanopore sequencing is a method under development since 1995 [1][2] for sequencing DNA (determining the order in which nucleotides occur on a strand of DNA).

A nanopore is simply a small hole with an internal diameter of the order of 1 nanometer. Certain porous transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a somewhat larger hole (several tens of nanometers) in a piece of silicon, and then gradually filling it in using ion-beam sculpting methods which results in a much smaller diameter hole: the nanopore. Graphene[3] is also being explored as a synthetic substrate for solid-state nanopores.

alpha-hemolysin pore (made up of 7 identical subunits in 7 colors) and 12-mer single-stranded DNA (in white) on the same scale to illustrate DNA effects on conductance when moving through a nanopore. Below is an orthogonal view of the same molecules.

The theory behind nanopore sequencing is that when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it, an electric current due to conduction of ions through the nanopore can be observed. The amount of current is very sensitive to the size and shape of the nanopore. If single nucleotides (bases), strands of DNA or other molecules pass through or near the nanopore, this can create a characteristic change in the magnitude of the current through the nanopore.

Background

DNA could be passed through the nanopore for various reasons. For example, electrophoresis might attract the DNA towards the nanopore, and it might eventually pass through it. Or, enzymes attached to the nanopore might guide DNA towards the nanopore. The scale of the nanopore means that the DNA may be forced through the hole as a long string, one base at a time, rather like threading through the eye of a needle. As it does so, each nucleotide on the DNA molecule may obstruct the nanopore to a different, characteristic degree. The amount of current which can pass through the nanopore at any given moment therefore varies depending on whether the nanopore is blocked by an A, a C, a G or a T or a section of DNA that includes more than one of these bases (kmer). The change in the current through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. Alternatively, a nanopore might be used to identify individual DNA bases as they pass through the nanopore in the correct order - this approach was published but not commercially developed by Oxford Nanopore Technologies and Professor Hagan Bayley.[4]

Using Nanopore sequencing, a single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labelling step or the need for optical instrumentation to identify the chemical label. The versatility of the nanopore concept is underlined by the fact that it has also been proposed for detection of life on other planets, since it is not necessarily restricted to the detection of the genetic information carrier DNA, but in general can be applied to sequence chain-like genetic information carriers without knowing the exact structure of their building blocks.[5] Nanopore-based DNA analysis techniques are being industrially developed by Oxford Nanopore Technologies (developing strand sequencing using protein nanopores, and solid-state sequencing through internal R&D and collaborations with academic institutions). As of June 2015, the MinION device from Oxford Nanopore has now been used by a number of participants in the MinION Access Programme. Publications on the method outline its use in rapid identification of viral pathogens,[6] monitoring ebola,[7] environmental monitoring,[8] food safety monitoring, monitoring of antibiotic resistance,[9] haplotyping[10] and other applications. Other companies have noted that they have Nanopore programmes, NabSys (using a library of DNA probes and using nanopores to detect where these probes have hybridized to single stranded DNA) and NobleGen (using nanopores in combination with fluorescent labels). IBM has noted research projects on computer simulations of translocation of a DNA strand through a solid-state nanopore, but not projects on identifying the DNA bases on that strand.

Types of nanopores

Alpha hemolysin

Alpha hemolysin (αHL), a nanopore from bacteria that causes lysis of red blood cells, has been studied for over 15 years.[2] To this point, studies have shown that all four bases can be identified using ionic current measured across the αHL pore.[11][12] The structure of αHL is advantageous to identify specific bases moving through the pore. The αHL pore is ~10 nm long, with two distinct 5 nm sections. The upper section consists of a larger, vestibule-like structure and the lower section consists of three possible recognition sites (R1, R2, R3), and is able to discriminate between each base.[11][12]

Sequencing using αHL has been developed through basic study and structural mutations, moving towards the sequencing of very long reads. Protein mutation of αHL has improved the detection abilities of the pore.[4] The next proposed step is to bind an exonuclease onto the αHL pore. The enzyme would periodically cleave single bases, enabling the pore to identify successive bases. Coupling an exonuclease to the biological pore would slow the translocation of the DNA through the pore, and increase the accuracy of data acquisition.

Notably, theorists have shown that sequencing via exonuclease enzymes as described here is not feasible.[13] This is mainly due to diffusion related effects imposing a limit on the capture probability of each nucleotide as it is cleaved. This results in a significant probability that a nucleotide is either not captured before it diffuses into the bulk or captured out of order, and therefore is not properly sequenced by the nanopore, leading to insertion and deletion errors. Therefore, major changes are needed to this method before it can be considered a viable strategy.

A recent study has pointed to the ability of αHL to detect nucleotides at two separate sites in the lower half of the pore.[14] The R1 and R2 sites enable each base to be monitored twice as it moves through the pore, creating 16 different measurable ionic current values instead of 4. This method improves upon the single read through the nanopore by doubling the sites that the sequence is read per nanopore.

MspA

Mycobacterium smegmatis porin A (MspA) is the second biological nanopore currently being investigated for DNA sequencing. The MspA pore has been identified as a potential improvement over αHL due to a more favorable structure.[15] The pore is described as a goblet with a thick rim and a diameter of 1.2 nm at the bottom of the pore.[16] A natural MspA, while favorable for DNA sequencing because of shape and diameter, has a negative core that prohibited single stranded DNA(ssDNA) translocation. The natural nanopore was modified to improve translocation by replacing three negatively charged aspartic acids with neutral asparagines.[17]

The electric current detection of nucleotides across the membrane has been shown to be tenfold more specific than αHL for identifying bases.[15] Utilizing this improved specificity, a group at the University of Washington has proposed using double stranded DNA (dsDNA) between each single stranded molecule to hold the base in the reading section of the pore.[15][17] The dsDNA would halt the base in the correct section of the pore and enable identification of the nucleotide. A recent grant has been awarded to a collaboration from UC Santa Cruz, the University of Washington, and Northeastern University to improve the base recognition of MspA using phi29 polymerase in conjunction with the pore.[18]

Fluorescence

An effective technique to determine a DNA sequence has been developed using solid state nanopores and fluorescence.[19] This fluorescence sequencing method converts each base into a characteristic representation of multiple nucleotides which bind to a fluorescent probe strand-forming dsDNA. With the two color system proposed, each base is identified by two separate fluorescences, and will therefore be converted into two specific sequences. Probes consist of a fluorophore and quencher at the start and end of each sequence, respectively. Each fluorophore will be extinguished by the quencher at the end of the preceding sequence. When the dsDNA is translocating through a solid state nanopore, the probe strand will be stripped off, and the upstream fluorophore will fluoresce.[19][20]

This sequencing method has a capacity of 50-250 bases per second per pore, and a four color fluorophore system (each base could be converted to one sequence instead of two), will sequence over than 500 bases per second.[19] Advantages of this method are based on the clear sequencing readout—using a camera instead of noisy current methods. However, the method does require sample preparation to convert each base into an expanded binary code before sequencing. Instead of one base being identified as it translocates through the pore, ~12 bases are required to find the sequence of one base.[19]

Electron tunneling

Measurement of electron tunneling through bases as ssDNA translocates through the nanopore is an improved solid state nanopore sequencing method. Most research has focused on proving bases could be determined using electron tunneling. These studies were conducted using a scanning probe microscope as the sensing electrode, and have proved that bases can be identified by specific tunneling currents.[21] After the proof of principle research, a functional system must be created to couple the solid state pore and sensing devices.

Researchers at the Harvard Nanopore group have engineered solid state pores with single walled carbon nanotubes across the diameter of the pore.[22] Arrays of pores are created and chemical vapor deposition is used to create nanotubes that grow across the array. Once a nanotube has grown across a pore, the diameter of the pore is adjusted to the desired size. Successful creation of a nanotube coupled with a pore is an important step towards identifying bases as the ssDNA translocates through the solid state pore.

Another method is the use of nanoelectrodes on either side of a pore.[23][24] The electrodes are specifically created to enable a solid state nanopore's formation between the two electrodes. This technology could be used to not only sense the bases but to help control base translocation speed and orientation.

Challenges

One challenge for the 'strand sequencing' method was in refining the method to improve its resolution to be able to detect single bases. In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change. This low resolution is because the DNA strand moves rapidly at the rate of 1 to 5μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. The problem is being tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies and Oxford Nanopore employs a 'kmer approach', analyzing more than one base at any one time so that stretches of DNA are subject to repeat interrogation as the strand moves through the nanopore one base at a time.[25] Various techniques including algorithmic have been used to improve the performance of the MinION technology since it was first made available to users. More recently effects of single bases due to secondary structure or released mononucleotides have been shown.[26][27] Professor Hagan Bayley, founder of Oxford Nanopore, proposed in 2010 that creating two recognition sites within an alpha hemolysin pore may confer advantages in base recognition.[28]

One challenge for the 'exonuclease approach', initially developed but not progressed by Oxford Nanopore Technologies,[29] where a processive enzyme feeds individual bases, in the correct order, into the nanopore, is to integrate the exonuclease and the nanopore detection systems. In particular,[30] the problem is that when an exonuclease hydrolyzes the phosphodiester bonds between nucleotides in DNA, the subsequently released nucleotide is not necessarily guaranteed to directly move in to, say, a nearby alpha-hemolysin nanopore. One idea[30] is to attach the exonuclease to the nanopore, perhaps through biotinylation to the beta barrel hemolsyin. The central pore of the protein may be lined with charged residues arranged so that the positive and negative charges appear on opposite sides of the pore. However, this mechanism is primarily discriminatory and does not constitute a mechanism to guide nucleotides down some particular path.

Commercialization

Agilent Laboratories was the first to license and develop nanopores [31] but does not have any current disclosed research in the area.

The company Oxford Nanopore Technologies in 2008 licensed technology from Harvard, UCSC and other universities [32] and is developing protein and solid state nanopore technology with the aim of sequencing DNA and identifying biomarkers, drugs of abuse and a range of other molecules. They revealed the idea of their first working device in February 2012.[33] The Company introduced its pocket-sized sensing device MinION to an early-access community in early 2014[34] and made it commercially available in May 2015. A variety of applications for the device have now been developed including environmental or pathogen monitoring, genome research, investigation of fetal DNA.[35] The higher-throughput PromethION will be available to early users through an Access Programme.[36]

Sequenom licensed nanopore technology from Harvard in 2007[37] using an approach that combines nanopores and fluorescent labels. This technology was subsequently licensed to Noblegen.[38]

NABsys was spun out of Brown University and is researching nanopores as a method of identifying areas of single stranded DNA that have been hybridized with specific DNA probes.

Genia applied for a patent[39] on a method of nanopore sequencing which uses a series of RNA "speed-bump" molecules to infer the sequence of the molecule based on the pattern of delayed progression.

References

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  2. 1 2 Kasianowicz, JJ; Brandin E; Branton D; Deamer DW (1996-11-26). "Characterization of individual polynucleotide molecules using a membrane channel". Proc Natl Acad Sci USA 93 (24): 13770–3. doi:10.1073/pnas.93.24.13770. PMC 19421. PMID 8943010.
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  7. ↑ Nick Loman (15 May 2015). "How a small backpack for fast genomic sequencing is helping combat Ebola". The Conversation.
  8. ↑ "TGAC's take on the first portable DNA sequencing 'laboratory'". EurekAlert!. 19 March 2015.
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  10. ↑ "Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes - F1000Research".
  11. 1 2 Stoddart D; Heron A; Mikhailova E; Maglia G; Bayley H (2009). "Single-nucleotide discrimination in immobilized DNA oglionucleoties with a biological nanopore". Proc. Natl. Acad. Sci. USA 106: 7702–7707. doi:10.1073/pnas.0901054106. PMC 2683137. PMID 19380741.
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  13. ↑ Reiner, Joseph E.; Balijepalli, Arvind; Robertson, Joseph W. F.; Drown, Bryon S.; Burden, Daniel L.; Kasianowicz, John J. (2012-12-07). "The effects of diffusion on an exonuclease/nanopore-based DNA sequencing engine". The Journal of Chemical Physics 137 (21): 214903. doi:10.1063/1.4766363. ISSN 0021-9606. PMC 4108639. PMID 23231259.
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  15. 1 2 3 Manrao, E; Derrington, I; Pavlenok, M; Niederweis, M; Gundlach, J (2011). "Nucleotide discrimination with DNA immobilized in the MspA nanopore". PLoS ONE 6: 10. doi:10.1371/journal.pone.0025723.
  16. ↑ Faller, M; et al. (2004). "The structure of a mycobacterial outer-membrane channel". Science 303: 1189–1192. doi:10.1126/science.1094114. PMID 14976314.
  17. 1 2 Butler, TZ; Pavlenok, M; Derrington, I; Niederweis, M; Gundlach, J (2008). "Single-molecule DNA detection with an engineered MspA protein nanopore". Proc. Natl. Acad. Sci. USA 106 (9): 20647–20652. doi:10.1073/pnas.0807514106.
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  19. 1 2 3 4 McNally, B; Singer, A; Yu, Z; Sun, Y; Weng, Z; Meller, A (2010). "Optical recognition of converted DNA nucleotides for single molecule DNA sequencing using nanopore arrays". Nano Lett. 10 (6): 2237–2244. doi:10.1021/nl1012147. PMC 2883017. PMID 20459065.
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  22. ↑ Sadki, ES; Garaj, S; Vlassarev, D; Golovchenko, JA; Branton, D (2011). "Embedding a carbon nanotube across the diameter of a solid state nanopore". J. Vac. Sci. Technol 29: 5.
  23. ↑ Ivanov, A; Instuli, E; McGilvery, C; Baldwin, G; McComb, D; Albrecht, T; Edel, J (2011). "DNA tunneling detector embedded in a nanopore". Nano Lett. 11: 279–285. doi:10.1021/nl103873a.
  24. ↑ "Archived copy". Archived from the original on November 29, 2011. Retrieved December 17, 2011.
  25. ↑ Hagan Bayley, Sequencing single molecules of DNA, Current Opinion in Chemical Biology, Volume 10, Issue 6, Model systems / Biopolymers, December 2006, Pages 628-637, ISSN 1367-5931, doi:10.1016/j.cbpa.2006.10.040. <http://www.sciencedirect.com/science/article/B6VRX-4MCWB5T-1/2/7090e8c122adfca35c1a2e99fa177f7c>
  26. ↑ Ashkenas, N; Sánchez-Quesada J; Bayley H; Ghadiri MR (2005-02-18). "Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores". Angew Chem Int Ed Engl 44 (9): 1401–4. doi:10.1002/anie.200462114. PMC 1828035. PMID 15666419.
  27. ↑ Winters-Hilt, S; Vercoutere W; DeGuzman VS; Deamer D; Akeson M; Haussler D (February 2003). "Highly accurate classification of Watson-Crick basepairs on termini of single DNA molecules". Biophys J. 84 (2 Pt 1): 967–76. doi:10.1016/S0006-3495(03)74913-3. PMC 1302674. PMID 12547778.
  28. ↑ Stoddart D; Maglia G; Mikhailova E; Heron A; Bayley H (2010). "Multiple Base-Recognition Sites in a Biological Nanopore: Two Heads are Better than One". Angew Chem Int Ed Engl 49 (3): 556–9. doi:10.1002/anie.200905483. PMC 3128935. PMID 20014084.
  29. ↑ Astier, Y; Braha O; Bayley H (2006-02-08). "Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter". J Am Chem Soc. 128 (5): 1705–10. doi:10.1021/ja057123+. PMID 16448145.
  30. 1 2 Rusk, Nicole (2009-04-01). "Cheap Third-Generation Sequencing". Nature Methods 6 (4): 244–245. doi:10.1038/nmeth0409-244a.
  31. ↑ "Agilent Laboratories, Harvard University Collaborate On Development of Breakthrough Technology for the Analysis of Nucleic Acids". Business Wire. 2001-05-21.
  32. ↑ "Harvard University And Oxford Nanopore Technologies Announce Licence Agreement To Advance Nanopore DNA Sequencing".
  33. ↑ "Nanopore genome sequencer makes its debut". Nature News & Comment.
  34. ↑ "The MinION device: a miniaturised sensing system - Oxford Nanopore Technologies".
  35. ↑ "Publications and more from the MAP community". Publications and more from the MAP community.
  36. ↑ "PEAP (PromethION Early Access Programme) - Community - Oxford Nanopore Technologies".
  37. ↑ "Sequenom to Develop Third-Generation Nanopore-Based Single Molecule Sequencing Technology".
  38. ↑ "Noblegen commercialise optical sequencing".
  39. ↑ "Patent US20130244340 - Nanopore Based Molecular Detection and Sequencing".

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