Everything You Want to Know about Oxford Nanopore
Over the last week, science blogs described the sequencing instrument of Oxford Nanopore as everything from hi-tech washing machine to Nigerian scam, ZX-81 and Turbo Pascal combined together. We emailed the CEO of this supposedly ultra-secretive company with those blog posts, and to our utter surprise, received a long email reply describing everything they accomplished over the last two years.
Well, we made up the last part in the above paragraph. No email exchange took place with their company (editing to clarify this point), but we learned a lot about their technologies. How so? Because unlike the public impression we get about them as being highly secretive, the company is actually very open and regularly posts many details about their technology online. From those postings, a knowledgeable person should be able to put together quite a bit about what they are up to and what their likely bottlenecks are. You do not need a password to access that site and simply click on the links below. The figures are not available from the linked website, but they can be easily found online without help from NSA.
1. Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
Summary of the Invention
The inventors have surprisingly demonstrated that both strands of a double stranded target polynucleotide can be sequenced by a nanopore when the two strands are linked by a bridging moiety and then separated. Furthermore, the inventors have also surprisingly shown that an enzyme, such as Phi29 DNA polymerase, is capable of separating the two strands of a double stranded polynucleotide, such as DNA, linked by a bridging moiety and controlling the movement of the resulting single stranded polynucleotide through the transmembrane pore.
The ability to sequence both strands of a double stranded polynucleotide by linking the two strands with a bridging moiety has a number of advantages, not least that both the sense and anti-sense strands of the polynucleotide can be sequenced. These advantages are discussed in more detail below.
Accordingly, the invention provides a method of sequencing a double stranded target polynucleotide, comprising:
(a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety;
(b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety;
(c) moving the single stranded polynucleotide through a transmembrane pore such that a proportion of the nucleotides in the single stranded polynucleotide interact with the pore; and
(d) measuring the current passing through the pore during each interaction and thereby determining or estimating the sequence of the target polynucleotide, wherein the separating in step (b) comprises contacting the construct with a
polynucleotide binding protein which separates the two strands of the target
polynucleotide.
The invention also provides:
a kit for preparing a double stranded target polynucleotide for sequencing comprising (a) a bridging moiety capable of linking the two strands of the target polynucleotide at or near one end and (b) at least one polymer;
a method of preparing a double stranded target polynucleotide for sequencing, comprising:
(a) linking the two strands of the target polynucleotide at or near one end with a bridging moiety; and
(b) attaching one polymer to one strand at the other end of the target polynucleotide and thereby forming a construct that allows the target polynucleotide to be sequenced using a transmembrane pore;
a method of sequencing a double stranded target polynucleotide, comprising:
(a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety; (b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety;
(c) synthesising a complement of the single stranded polynucleotide, such that the single stranded polynucleotide and complement form a double stranded
polynucleotide;
(d) linking the two strands of the double stranded polynucleotide at or near one end of the double stranded polynucleotide using a bridging moiety;
(e) separating the two strands of the double stranded polynucleotide to provide a further single stranded polynucleotide comprising the original single stranded polynucleotide linked to the complement by the bridging moiety;
(f) moving the complement through a transmembrane pore such that a proportion of the nucleotides in the complement interact with the pore; and
(g) measuring the current passing through the pore during each interaction and thereby determining or estimating the sequence of the target polynucleotide, wherein the separating in step (e) comprises contacting the construct with a polynucleotide binding protein which separates the two strands of the target polynucleotide;
an apparatus for sequencing a double stranded target polynucleotide, comprising: (a) a membrane; (b) a plurality of transmembrane pores in the membrane; (c) a plurality of polynucleotide binding proteins which are capable of separating the two strands of the target polynucleotide; and (d) instructions for carrying out the method of the invention; and
an apparatus for sequencing a double stranded target polynucleotide, comprising: (a) a membrane; (b) a plurality of transmembrane pores in the membrane; and (c) a plurality of polynucleotide binding proteins which are capable of separating the two strands of the target polynucleotide, wherein the apparatus is set up to carry out the method of the invention.. Description of the Figures
Fig. 1 shows a schematic of enzyme controlled dsDNA and ssDNA translocation through a nanopore. An enzyme (e.g. Phi29 DNA polymerase) that is incubated with dsDNA having an ssDNA leader binds at the ssDNA-dsDNA interface. DNA- enzyme complexes are captured by a nanopore under an applied field. Under the field, the template strand of the DNA is slowly stripped through the enzyme in a controlled base-by-base manner, in the process unzipping the complementary primer strand of the dsDNA in or above the enzyme. Once the enzyme reaches the end of the dsDNA it falls off the D A, releasing it through the nanopore.
Fig. 2 shows another schematic of enzyme controlled dsDNA and ssDNA translocation through a nanopore. The dsDNA has a hairpin turn linking the sense and anti-sense strands of the dsDNA. Once the enzyme reaches the hairpin it remains bound to the DNA, proceeds around the hairpin turn, and along the anti-sense strand. In the hairpin and antisense regions the enzyme functions as an ssDNA molecular brake, continuing to sufficiently control translocation of the DNA through the nanopore to sequence the DNA.
Fig. 3 shows a schematic overview of reading around a hairpin of dsDNA using the ability of the enzyme to control movement in ssDNA regions. The dsDNA has a 5’-ssDNA leader to allow capture by the nanopore. This is followed by a dsDNA section, where the sense and anti-sense strands are connected by a hairpin. The hairpin can optionally contain markers (e.g. abasic residues, shown in Fig. 3 as a cross) that are observed during sequencing, which permit simple identification of the sense and anti-sense strands during sequencing. The 3 ‘-end of the anti-sense strand can optionally also have a 3’-ssDNA overhang, which if greater than ~20 bases allows full reading of the anti- sense strand (the read-head of the nanopore is -20 bases downstrand from the top of the DNA in the enzyme).
Fig. 4 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 marks the anti-sense section. This figure shows DNA sequencing of a short dsDNA construct. In this construct the dsDNA section is not connected by a hairpin, so the enzyme falls off the end of the DNA, and only the template/sense strand is sequenced (except for the last -20 bases).
Fig. 5 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 the anti-sense section. DNA sequencing of a short dsDNA construct with a hairpin. In this construct the enzyme moves along the sense strand, around the hairpin loop, and down the anti- sense strand, permitting sequencing of both the sense and the first part of the anti-sense strand.
Fig. 6 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 the anti-sense section. Similar to Fig. 5, this construct permits sequencing of both the sense and anti-sense strands, but the additional 3’-ssDNA overhang permits reading of the full length of the anti- sense strand before the enzyme falls off the end of the DNA. Fig. 7 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 the anti-sense section. Similar to Fig. 5, this construct permits sequencing of both the sense and anti-sense strands, however, this construct has a single abasic residue (shown as a cross) in the hairpin, which provides a clear marker in the DNA sequence to identify the sense and anti- sense sections.
Fig. 8 shows the consensus DNA sequence of UA02 through MspA. Section 1 marks the homopolymeric 5 ‘-overhang initially in the nanopore. Section 2 marks the sense section of the DNA strand. Section 3 marks the turn. Section 4 marks the anti-sense region of the DNA strand. The polynucleotide sequence that corresponds to each section is shown below that section number.
Fig. 9 shows a schematic of a genomic template for unzipping through MspA nanopores using Phi29 DNA polymerase. It shows a general design outline for creating dsDNA suitable for reading around hairpins. The constructs have a leader sequence with optional marker (e.g. abasic DNA) for capture in the nanopore, and hairpin with optional marker, and a tail for extended reading into anti-sense strand with optional marker.
Fig. 10 shows a schematic of the adapter design for ligating ssDNA overhangs (left) and hairpin turns (right) onto genomic dsDNA. X = abasic DNA. Choi = cholesterol-TEG DNA modification.
Fig. 11 shows typical polymerase controlled DNA movement of a 400mer-hairpin through MspA using Phi29 DNA polymerase. Sense region = abasic 1 to 2. Anti- sense region = abasic 2 to 3.
Fig. 12 shows a consensus DNA sequence profile from multiple polymerase controlled DNA movements of a 400mer-hairpin through MspA. Sense region = abasic 1 to 2. Anti-sense region = abasic 2 to 3.
Fig. 13 shows a schematic of an alternative sample preparation for sequencing. A construct is illustrated comprising the target polynucleotide and a bridging moiety (hairpin) linking the two strands of the target polynucleotide. The construct also comprises a leader polymer (a single stranded sequence), a tail polymer (also a single stranded sequence) and an abasic marker region within the leader. The marker may prevent the enzyme from making the template completely blunt ended i.e. filling in opposite the required leader ssDNA. A strand displacing polymerase (nucleic acid binding protein) separates the two strands of the construct, initiating either via a complementary primer or by protein primed amplification from the tail polymer. A complement is generated to the resulting single stranded polynucleotide. The complement and the original sense and antisense single stranded polynucleotide analyte can be further modified by addition of a second bridging moiety (hairpin).
Fig. 14 shows a specific preparation of the construct comprising the target
polynucleotide.
Fig. 15 shows where amplification may be added as part of the sample preparation to aid the detection of epigenetic information. A nucleotide has been constructed so that the following information is read through the pore: sense (original), antisense (original), bridging moiety, sense (replicate), antisense (replicate). Information on the methylated base (mC) is therefore obtained four times.
Fig. 16 shows how RNA can be sequenced. A bridging moiety is attached to a piece of
R A and the DNA reverse complement added to the RNA via a reverse transcriptase. The RNA is read, followed by the DNA of the reverse complement.
Fig. 17 shows a schematic of helicase controlled dsDNA and ssDNA translocation through a nanopore. The dsDNA has a hairpin turn linking the sense and anti- sense strands of the dsDNA. Once the enzyme reaches the hairpin it remains bound to the DNA, proceeds around the hairpin turn, and along the anti-sense strand. In the hairpin and antisense regions the enzyme functions as an ssDNA molecular brake, continuing to sufficiently control translocation of the DNA through the nanopore to sequence the DNA.
Fig. 18 shows the polynucleotide MONO hairpin construct (SEQ ID NOs: 29 to 35) used in Example 4.
Fig. 19 shows a typical helicase controlled DNA movement of a 400 bp hairpin (SEQ ID NOs: 29 to 35 connected as shown in Fig. 18) through an MspA nanopore (MS(B1-G75S-G77S- L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). Sense = region 1. Anti-sense = region 2.
Fig. 20 shows the beginning of a typical helicase controlled DNA movement of a 400 bp hairpin (SEQ ID NOs: 29 to 35 connected as shown in Fig. 18) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations
G75S/G77S/L88N/Q126R)). The polyT region at the beginning of the sequence is highlighted with a * and the abasic DNA bases as a #.
Fig. 21 shows the transition between the sense and antisense regions of a typical helicase controlled DNA movement of a 400 bp-hairpin (SEQ ID NOs: 29 to 35 connected as shown in Fig. 18) through an MspA nanopore (MS(B1-G75S- G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). The transition region between the sense and antisense regions of the sequence is highlighted by a * , the sense region labeled 1 and the antisense region labeled 2. Fig. 22 shows an example sample prep method for forming DUO hairpin constructs. The double stranded DNA analyte is contacted by and modified to contain a Y-shaped adapter (the sense strand (SEQ ID NO: 29 attached to SEQ ID NO: 30 via four abasic DNA bases) of this adaptor contains the 5’ leader, a sequence that is complementary to the tether (SEQ ID NO: 35, which at the 3’ end of the sequence has six iSpl 8 spacers attached to two thymine residues and a 3’ cholesterol TEG) and 4 abasics and the antisense half of the adaptor contains a 3’ hairpin (SEQ ID NO: 31)) at one end of the duplex and a hairpin (SEQ ID NO: 32) at the other. The Y- shaped adapter itself also carries a 3 ‘-hairpin (SEQ ID NO: 31), which allows extension either by a polymerase or by ligation. This extension is preferentially carried out by a mesophilic polymerase that has strand displacement activity. As the polymerase extends from the 3 ‘ of the Y-shaped adapter hairpin (SEQ ID NO: 31) it copies the antisense strand (SEQ ID NO: 34) and so displaces the original sense strand (SEQ ID NO: 33). When the polymerase reaches the end of the antisense strand (SEQ ID NO: 34) it fills-in opposite the hairpin (SEQ ID NO: 32) and then begins to fill-in opposite the now single stranded and original sense strand (SEQ ID NO: 33). Extension is then halted by a section of abasic or spacer modifications (other possible modifications which could halt enzyme extension include RNA, PNA or morpholino bases and iso-dC or iso- dG) to leave the 5 ‘-region of the Y-shaped adapter single stranded (SEQ ID NO: 29).
Fig. 23 shows the specific preparation method used in Example 5 for preparing a DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25). A -400 bp region of PhiX 174 was PCR amplified with primers containing Sad and Kpnl restriction sites (SEQ ID Nos: 27 and 28 respectively). Purified PCR product was then Sad and Kpnl digested before aY- shaped adapter (sense strand sequence (SEQ ID NO: 29 attached to SEQ ID NO: 30 via four abasic DNA bases) is ligated onto the 5’ end of SEQ ID NO: 33 and the anti-sense strand (SEQ ID NO: 31) is ligated onto the 3 ‘ end of the SEQ ID NO: 34) and a hairpin (SEQ ID NO: 32, used to join SEQ ID NO’s: 33 and 34) were ligated to either end, using T4 DNA ligase (See Fig. 18 for final DNA construct). The doubly ligated product was PAGE purified before addition of lenow DNA polymerase, SSB and nucleotides to allow extension from the Y-shaped adapter hairpin (SEQ ID NO: 31). To screen for successful DUO product a series of mismatch restriction sites were incorporated into the adapter sequences, whereby the enzyme will cut the analyte only if the restriction site has been successfully replicated by the DUO extension process.
Fig. 24 shows that the adapter modified analyte (MONO, SEQ ID NOs: 29-35 connected as shown in Fog. 18) in the absence of polymerase does not digest with the restriction enzymes (see gel on the left, Key: M = Mfel, A = Agel, X = Xmal, N = NgoMIV, B = BspEl), due to the fact they are mismatched to one another. However, on incubation with polymerase there is a noticeable size shift and the shifted product (DUO, SEQ ID NOs: 29-36 connected as shown in Fig. 25) now digests as expected with each of the restriction enzymes (see gel on the right, Key: M = Mfel, A = Agel, X = Xma\, N = NgoMIV, B = BspEl).
Fig. 25 shows the polynucleotide DUO hairpin construct (SEQ ID NOs: 29 to 36) used in Examples 6.
Fig. 26 shows two typical helicase controlled DNA movements for the DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations
G75S/G77S/L88N/Q126R)). Sense original = region 1. Anti-sense original = region 2. Sense replicate = region 3. Anti-sense replicate = region 4.
Fig. 27 shows an expanded view of a typical helicase controlled DNA movement for the DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). Sense original = region 1. Anti- sense original = region 2. Sense replicate = region 3. Anti-sense replicate = region 4.
Fig. 28 shows an expanded view of a typical transition between the sense original and antisense original regions of the DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25) when under helicase controlled DNA movement through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations.
An apparatus for supporting an array of layers of amphiphilic molecules, the apparatus comprising: a body,formed in a surface of the body, an array of sensor wells capable of supporting a layer of amphiphilic molecules across the sensor wells, the sensor wells each containing an electrode for connection to an electrical circuit, and formed in the surface of the body between the sensor wells, flow control wells capable of smoothing the flow of a fluid across the surface.
The invention relates to a new method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or absence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores.
4. Analysis of measurements of a polymer
A time-ordered series of measurements of a polymer made during translocation of the polymer through a nanopore are analysed. The measurements are dependent on the identity of k-mers in the nanopore, a k-mer being k polymer units of the polymer, where k is a positive integer. The method involves deriving, from the series of measurements, a feature vector of time-ordered features representing characteristics of the measurements; and determining similarity between the derived feature vector and at least one other feature vector.
5. Method for characterising a polynucelotide by using a xpd helicase
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and an XPD helicase. The helicase controls the movement of the target polynucleotide through the pore.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.
7. Analysis of a polymer comprising polymer units
A sequence of polymer units in a polymer (3), eg. DNA, is estimated from at least one series of measurements related to the polymer, eg. ion current as a function of translocation through a nanopore (1), wherein the value of each measurement is dependent on a k-mer being a group of k polymer units (4). A probabilistic model, especially a hidden Markov model (HMM), is provided, comprising, for a set of possible k-mers: transition weightings representing the chances of transitions from origin k-mers to destination k-mers; and emission weightings in respect of each k-mer that represent the chances of observing given values of measurements for that k-mer. The series of measurements is analysed using an analytical technique, eg. Viterbi decoding, that refers to the model and estimates at least one estimated sequence of polymer units in the polymer based on the likelihood predicted by the model of the series of measurements being produced by sequences of polymer units. In a further embodiment, different voltages are applied across the nanopore during translocation in order to improve the resolution of polymer units.
8. Piston seal
A piston head for a syringe pump comprises a barrier portion for driving fluid through a syringe pump barrel, wherein a peripheral section of the barrier portion is shaped to seal against the syringe pump barrel; and a resilient member arranged to resist deformation of the shaped peripheral section of the barrier portion.
How about data? We have not seen any nucleotide-level data yet, but the first patent mentioned above contains many figures showing their electrical signals. One informative comment at OmicsOmics blog speculated on what could be going on at the company.
Interesting discussion here. I too went ahead and glanced through OxNano’s patent applications. Based on my very quick review, here is what I found
1. They have done extensive amount of work on mspA, but Illumina licenced the pore now. Not sure what is going on there
2. The closest I saw anything related to sequencing was a bioinformatics patent application that describes how to convert K mer signatures to sequence
- there was’t any sequencing data in that application
3. They seem to have some cool motors. Helicases from some extremophiles
4. One new pore called lysenin, not sure how it compares to mspA. No sequencing information in the patent application
5. Some algorithms to build consensus among traces (squiggles, if that is the terminology we are using)
6. There was one application where the did some algorithmic learning on a known piece of DNA, I didn’t see that extended to an unknown sample
Regarding the comment that investors probably saw sequencing data, that could be true. Wonder if that caused the rift with Illumina. They clearly convinced Illumina with a sequencing scheme, now they have convinced other investors of a different scheme. Time to convince the users.
Our views are in line with another comment -
The squiggles have been around for quite some time now. Both the Akeson and Gundlach labs have published the squiggles. Credit to Oxford for putting things in a nice package, but unless I see them convert the squiggles to sequence, I, sadly, will have to assume they are being evasive and just trying to buy time.
…
This other thought, I keep having because I hear how secretive and paranoid Oxford are. I am beginning to wonder if it is just a rouse to hide serious deficiencies they are trying, or unable to fix (with lots of investors and the world looking at them).
Buy time is what it is.
