Multi-pass sequencing

Patent metadata

Field	Value
Publication number	US-2018282800-A1
Application number	US-201515525253-A
Country	US
Kind code	A1
Filing date	Nov 10, 2015
Priority date	Nov 11, 2014
Publication date	Oct 4, 2018
Grant date	—

Abstract

Improved single molecule sequencing methods, compositions, and devices, are provided. In a first aspect, the present invention provides a multi-pass method of sequencing a target sequence using nanopore sequencing, the method comprising: i) providing a non-naturally occurring concatemer nucleic acid molecule comprising a plurality of copies of the target sequence; ii) nanopore sequencing at least three copies of the target sequence in the concatemer, thereby obtaining a multi-pass sequence dataset, wherein the multi-pass sequence dataset comprises target sequence datasets for the at least three copies of the target sequence; and iii) using the multi-pass sequence dataset to determine the target sequence.

First claim

1 . A multi-pass method of sequencing a target sequence using nanopore sequencing, the method comprising:
i) providing a non-naturally occurring concatemer nucleic acid molecule comprising a plurality of copies of the target sequence; ii) nanopore sequencing at least three copies of the target sequence in the concatemer, thereby obtaining a multi-pass sequence dataset, wherein the multi-pass sequence dataset comprises target sequence datasets for the at least three copies of the target sequence; and iii) using the multi-pass sequence dataset to determine the target sequence.

2 . The method of claim 1 , wherein adjacent copies of the target sequence are separated by a non-target sync sequence with a predetermined sequence and the nanopore sequencing further comprises sequencing at least four sync sequences.

3 . The method of claim 2 , wherein all of the sync sequences are the same.

4 . The method of claim 2 , wherein the sync sequences are shorter than the target sequence.

5 . The method of claim 2 , wherein the method comprises using the sync sequences to align the target sequence datasets.

6 . The method of claim 1 , wherein the multi-pass sequence has an accuracy of at least Q6.

7 . The method of claim 1 , wherein the concatemer comprises at least at least 5 copies, optionally at least 8 copies, of the target sequence.

8 . The method of claim 1 , wherein the concatemer comprises a calibration sequence.

9 . The method of claim 1 , wherein the providing the concatemer comprises circularizing a nucleic acid fragment comprising the target sequence to product a circular nucleic acid; and
using the circular nucleic acid as a template for rolling circle replication, thereby providing a concatemer.

10 . A nanopore sequencing method with single nanopore resolution calibration comprising:
i) providing a non-naturally occurring nucleic acid molecule comprising a calibration sequence and a target sequence; ii) nanopore sequencing the nucleic acid molecule, thereby obtaining calibration information and target sequence information; and iii) using the calibration information and the target sequence information to determine the target sequence.

11 . The method of claim 10 , wherein step iii) further comprises generating a nanopore specific base call model from the calibration information and applying the nanopore specific base call model to the target sequence information.

12 . The method of claim 11 , wherein the method further comprises nanopore sequencing a plurality of copies of the nucleic acid molecule with a plurality of nanopores.

13 . The method of claim 11 , wherein the method further comprises nanopore sequencing a plurality of copies of the target sequence with a single nanopore.

14 . The method of claim 13 , wherein the method comprises providing a non-naturally occurring concatemer nucleic acid molecule containing the plurality of copies of the target sequence.

15 .- 23 . (canceled)

24 . An array of nanopores, wherein each nanopore of the array is physically associated with a concatemer nucleic acid, wherein the concatemer nucleic acid comprises a plurality of copies of a target sequence and a plurality of non-target sync sequences having a predetermined sequence, and wherein the non-target sync sequences are positioned between adjacent copies of the target sequence.

25 . The array of claim 24 , wherein each nanopore of the array is physically associated with a concatemer nucleic acid containing a structurally different target sequence.

26 . A massively-parallel, randomly-distributed, nanopore sequencing device comprising:
a surface comprising a plurality of randomly distributed nanopores, wherein at least 10% of the randomly distributed nanopores are operably linked to a unique electrode.

27 . The nanopore sequencing device of claim 26 , wherein fewer than 50% of the nanopores are operably linked to a unique electrode.

28 . The device of claim 27 , wherein at least 100 million nanopores are operably linked to a unique electrode.

29 . The device of claim 26 , wherein the device comprises an ordered array of electrodes, each electrode configured to be capable of operably linking with a nanopore.

Assignees

• Bgi Shenzhen
• Bgi Shenzhen Co Ltd

Inventors

• Li Handong
• Tang Y Tom
• Yu Jing
• Jiang Hui
• Zhang Wenwei
• Fan Guangyi
• Zhang He
• Ma Kailong
• Geng Chunyu

Classifications

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title