EP-4737587-A1 - NANOPORE SEQUENCING METHOD USING CLOSER

Abstract

Provided in the present invention is a nanopore sequencing method using a closer. The method of the present invention enables a sequencing library to only bind within the nanopore capture range, ensuring that the library is efficiently captured by the nanopores during sequencing, thus improving the utilization efficiency of the library.

Inventors

• CHEN, Junyi
• JI, Zhouxiang
• ZENG, TAO
• LI, YuXiang
• DONG, Yuliang
• ZHANG, WENWEI
• XU, XUN

Assignees

• BGI Hangzhou CycloneSEQ Technoloy Co., Ltd.

Dates

Publication Date

20260506

Application Date

20230630

Claims (20)

1. A nanopore sequencing method, comprising the following steps: i. incubating a blocker with a membrane embedded with nanopores, such that the blocker is coupled to the membrane, wherein the blocker comprises: a tethering sequence, comprising a portion coupled to the membrane; a first strand, comprising a first sequence, and a second sequence at least partially complementary to a second strand; and the second strand, comprising a third sequence at least partially complementary to the first strand, configured to bind with a sequencing library by means of complementation with a sequencing adapter, and a fourth sequence at least partially complementary to the tethering sequence, ii. applying an electric field force to the membrane of step i, such that the first strand of the blocker within a nanopore capture range translocates through the nanopore, exposing the third sequence in the second strand of the blocker; and iii. binding, the third sequence, with the sequencing library via the sequencing adapter, such that the sequencing library is bound within the nanopore capture range and further captured by the nanopore, thereby achieving a sequencing on a strand to be sequenced ligated with the sequencing adapter.
2. The method according to claim 1, wherein the portion coupled to the membrane in the tethering sequence is a hydrophobic molecule, preferably, the hydrophobic molecule is selected from any one or more of: a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein and/or amino acid; more preferably, the hydrophobic molecule is selected from any one or more of: cholesterol, palmitate or tocopherol.
3. The method according to claim 1 or 2, wherein the first sequence consists of 10 to 50 nucleotides, iSpC3, iSp18, or a combination thereof.
4. The method according to any one of claims 1 to 3, wherein the first strand further comprises a nucleic-acid-controlling-protein binding sequence, and a first nucleic-acid-controlling-protein is introduced in step i.
5. The method according to claim 4, wherein the first nucleic-acid-controlling-protein is selected from any one of a helicase, topoisomerase, ligase, or polymerase.
6. The method according to claim 4 or 5, wherein the first nucleic-acid-controlling-protein acts in a direction from 3' to 5'or from 5' to 3'.
7. The method according to any one of claims 4 to 6, wherein the first strand further comprises a blocking sequence, located between the second sequence and the nucleic-acid-controlling-protein binding sequence.
8. The method according to claim 7, wherein the blocking sequence consists of at least one of iSp18, iSp9, iSpC3, iSpC6, and iSpC12.
9. The method according to claim 7 or 8, wherein the blocking sequence has a length of at least 1 bp.
10. The method according to any one of claims 1 to 9, wherein the second sequence, the third sequence, and the fourth sequence each has a length of at least 2 bp, preferably, the second sequence, the third sequence, and the fourth sequence each has a length of 5 bp to 30 bp.
11. The method according to any one of claims 1 to 10, wherein the third sequence is complementary, in a length of at least 2 bp, to the sequencing adapter of the sequencing library.
12. The method according to any one of claims 1 to 11, wherein the blocker is composed of DNA, RNA, LNA, PNA, BNA, or any combination thereof.
13. The method according to any one of claims 1 to 12, wherein the nanopore is a transmembrane protein pore or a solid-state pore, preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein, InvG, GspD, or any combination thereof.
14. The method according to any one of claims 1 to 13, wherein the membrane is an amphiphilic membrane, a high-molecular polymer membrane, or any combination thereof.
15. The method according to any one of claims 1 to 14, wherein a voltage applied during sequencing is above 10 mV, preferably, the voltage is 50 mV to 250 mV.
16. The method according to any one of claims 1 to 15, wherein the sequencing library is introduced into a reaction system simultaneously with, prior to, or subsequent to an introduction of the blocker.
17. The method according to any one of claims 1 to 16, wherein the sequencing adapter consists of two nucleic acid chains, with a side of one nucleic acid chain complementary to a side of the other nucleic acid chain, and another side of one nucleic acid chain complementary to another side of the other nucleic acid chain, the blocker binds to the sequencing adapter in a manner comprises: a. under the sequencing adapter having a Y-shaped structure, with a side being a non-complementary, single stranded portion and another side being a complementary, double stranded portion, the third sequence of the blocker complementarily binds to the single stranded portion of the sequencing adapter; or b. under the sequencing adapter having a side being a non-complementary, single stranded portion and another side being a complementary, double stranded portion comprising a RNA fragment, after degrading the RNA fragment with a ribonuclease, the third sequence of the blocker complementarily binds to a complementary sequence of the RNA fragment.
18. The method according to any one of claims 1 to 17, wherein the sequencing adapter binds with a second nucleic-acid-controlling-protein, forming a sequencing adapter complex, preferably, the second nucleic-acid-controlling-protein is a helicase.
19. The method according to any one of claims 1 to 18, wherein in step iii, the third sequence of the blocker is complementary to the sequencing adapter of the sequencing library, such that the blocker links to the sequencing library; and under an action of the electric field force, the strand to be sequenced in the sequencing library translocates through the nanopore, and a current signal change generated by translocation of the strand to be sequenced through the nanopore is detected, thereby obtaining sequence information of the strand to be sequenced.
20. A nanopore sequencing kit, comprising: a blocker, a sequencing buffer, a sequencing adapter and a second nucleic-acid-controlling-protein, wherein the blocker comprises: a tethering sequence, comprising a portion coupled to a membrane in the sequencing; a first strand, comprising a first sequence, and a second sequence at least partially complementary to a second strand; and the second strand, comprising a third sequence at least partially complementary to the first strand, and a fourth sequence at least partially complementary to the tethering sequence, wherein the sequencing adapter forms, with the second nucleic-acid-controlling-protein, a sequencing adapter complex to capture a strand to be sequenced, thereby producing a sequencing library, and the third sequence is configured to bind with the sequencing library complementarily, such that the sequencing library is bound within a nanopore capture range and further captured by the nanopore, to achieve sequencing.

Description

FIELD The present disclosure relates to the technical field of nanopore sequencing, and specifically to a nanopore sequencing method using a blocker. BACKGROUND Nanopore sequencing is a single-molecule detection technique featuring advantages such as high sequencing speed, long read length, direct sequencing capability, high throughput, low cost, small size and portability. During nanopore sequencing, a single nanopore is embedded in an insulating and impermeable membrane to form a stable ionic current channel. The sequencing library is coupled to the insulating and impermeable membrane under the action of a tethering sequence. Driven by voltage, the sequencing library within a nanopore capture range translocates through the nanopore in the form of single-stranded nucleic acid molecules under the control of motor proteins, reducing the ionic current flowing through the nanopore. Due to the differences in molecular structure and volume among different bases on the single-stranded nucleic acid molecule, the current flowing through the nanopore exhibits variations corresponding to the base sequence. Using algorithms to analyze the current variation signals, the sequence of the translocated single-stranded nucleic acid can be read in real time. As developed by Oxford Nanopore Technologies (ONT), the library to be sequenced is coupled to the insulating and impermeable membrane under the action of a tethering sequence (Fig. 1), and the specific process is as follows: i. the library to be sequenced binds to the tethering sequence; ii. after incubation, the library to be sequenced is coupled to the insulating and impermeable membrane embedded with nanopores under the action of the tethering sequence; iii. upon application of an electric field force, the library to be sequenced coupled within a nanopore capture range is captured for sequencing (see Patent CN103733063B). However, when coupling to the insulating and impermeable membrane embedded with nanopores under the action of the tethering sequence, the sequencing library will also bind to such areas in the insulating and impermeable membrane that nanopores are not successfully embedded, or even to residual membrane materials in the sequencing system. However, only a small fraction of the sequencing library that is bound within the nanopore capture range can be captured by the nanopores and sequenced, leading to substantial waste of the library due to its failed sequencing. Developing a method enabling the sequencing library to be bound within the nanopore capture range in a concentrated manner can ensure efficient capture to the library by nanopores during sequencing and improve the utilization efficiency of the library. A method developed by ONT for concentrating tethering complexes in an amphiphilic membrane region is illustrated in Fig. 2. The amphiphilic membrane is composed of multiple amphiphilic molecules and can be chemically and/or physically divided into a first region and a second region. Detectors, such as nanopores, are embedded in the first region. The tethering complex contains one or more hydrophilic components connected by a hydrophobic linker, and the tethering complex preferentially localises to the first region. The sequencing library can specifically bind to the hydrophilic components of the tethering complex, thereby binding the sequencing library to the first region (see Patent application CN115004030A). Qitan Technology Co., Ltd. Beijing has also developed a similar method (see Patent CN113999291B). However, the method developed by ONT for concentrating tethering complexes in the amphiphilic membrane region includes the following drawbacks: i. although the amphiphilic membrane is chemically and/or physically divided into the first region and the second region, and the tethering complex preferentially localises to the first region, the first region does not necessarily completely overlap with the nanopore capture range, there is a portion of the sequencing library bound to the tethering complex still failing to be sequenced thus being wasted; ii. in a large-scale array sequencing system developed by ONT, not all amphiphilic membranes are embedded with nanopores capable of valid sequencing, or the nanopores that were initially functional may lose their activity after a period of sequencing; nevertheless, such amphiphilic membranes are still positioned with the tethering complexes in the first region, resulting in the waste of sequencing libraries bound to these tethering complexes due to failed sequencing; iii. the tethering complex is composed of one or more hydrophilic components connected by a hydrophobic linker, featuring a complex structure, which leads to high difficulty and cost in synthesis and purification; iv. a large number of hydrophobic linkers of the tethering complexes penetrate the amphiphilic membrane, which reduces the stability of the amphiphilic membrane and negatively affects the service life of the sequencing system; an