Methods and Systems for Processing Polynucleotides

Patent metadata

Field	Value
Publication number	US-2019024166-A1
Application number	US-201816138448-A
Country	US
Kind code	A1
Filing date	Sep 21, 2018
Priority date	Jun 26, 2014
Publication date	Jan 24, 2019
Grant date	—

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

First claim

1 . A method for nucleic acid sequencing, comprising:
(a) co-partitioning a plurality of beads and a plurality of primers in a plurality of droplets, wherein a droplet of said plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, (ii) a primer from said plurality of primers, and (iii) a bead from said plurality of beads, wherein said bead comprises a nucleic acid barcode molecule coupled thereto, wherein said nucleic acid barcode molecule comprises a barcode sequence; (b) hybridizing said primer to a region at a 3′ end of said RNA molecule; (c) using an enzyme to extend said primer to generate a nucleic acid product comprising a sequence corresponding to said nucleic acid sequence of said RNA molecule, wherein said enzyme incorporates a sequence at a 3′ end of said nucleic acid product that is complementary to said nucleic acid barcode molecule; (d) hybridizing said nucleic acid barcode molecule to said nucleic acid product generated in (c) and extending said nucleic acid product using said nucleic acid barcode molecule as template, to generate a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, (1) said sequence corresponding to said nucleic acid sequence of said RNA molecule and (2) a complement of said barcode sequence; and (e) sequencing said barcoded nucleic acid molecule or a derivative thereof, wherein, after (a), said nucleic acid barcode molecule is released from said bead.

2 . The method of claim 1 , wherein said RNA molecule is from a cell.

3 . The method of claim 2 , wherein said droplet comprises said cell.

4 . The method of claim 3 , further comprising releasing said RNA molecule from said cell prior to (b).

5 . The method of claim 1 , wherein said bead further comprises a plurality of nucleic acid molecules coupled thereto.

6 . The method of claim 5 , wherein each of said plurality of nucleic acid molecules comprises said barcode sequence.

7 . The method of claim 6 , wherein each of said plurality of nucleic acid molecules comprises an additional barcode sequence that varies across said plurality of nucleic acid molecules.

8 . The method of claim 1 , further comprising, prior to (e), subjecting said barcoded nucleic acid molecule or derivative thereof to nucleic acid amplification.

9 . The method of claim 8 , wherein said nucleic acid amplification is performed subsequent to releasing said barcoded nucleic acid molecule or derivative thereof from said droplet.

10 . The method of claim 8 , wherein said nucleic acid amplification is polymerase chain reaction.

11 . The method of claim 1 , wherein said RNA molecule is a messenger ribonucleic acid (mRNA) molecule.

12 . The method of claim 1 , wherein in (a) said droplet comprises (i) an additional nucleic acid molecule comprising an additional nucleic acid sequence, and (ii) an additional nucleic acid barcode molecule comprising an additional barcode sequence, and wherein said additional nucleic acid molecule and said additional nucleic acid barcode molecule are used to generate an additional barcoded nucleic acid molecule comprising, from a 5′end to a 3′end, (1) said additional barcode sequence and (2) an additional sequence corresponding to said additional nucleic acid sequence.

13 . The method of claim 12 , wherein said additional nucleic acid barcode molecule is coupled to said bead.

14 . The method of claim 1 , wherein (b)-(d) are performed in said droplet.

15 . The method of claim 1 , further comprising releasing said barcoded nucleic acid molecule or a derivative thereof from said droplet.

16 . The method of claim 1 , wherein said barcoded nucleic acid molecule further comprises, towards a 5′ end, a functional sequence for permitting said barcoded nucleic acid molecule or a derivative thereof to couple to a flow cell of a sequencer.

17 . The method of claim 1 , wherein said sequence corresponding to said nucleic acid sequence of said RNA molecule is a reverse complement of said nucleic acid sequence.

18 . The method of claim 1 , further comprising, prior to (e), using said barcoded nucleic acid molecule or a derivative thereof and a pair of primers to generate barcoded nucleic acid molecules having a target nucleic acid sequence.

19 . The method of claim 18 , wherein said barcoded nucleic acid molecules having said target nucleic acid sequence or derivatives thereof are sequenced in (e).

20 . (canceled)

21 . The method of claim 1 , wherein said nucleic acid barcode molecule is released from said bead before said barcoded nucleic acid molecule is generated.

22 . The method of claim 1 , wherein said nucleic acid barcode molecule is released from said bead while said barcoded nucleic acid molecule is generated.

23 . The method of claim 1 , wherein said nucleic acid barcode molecule is released from said bead after said barcoded nucleic acid molecule is generated.

24 . The method of claim 1 , wherein said bead is a gel bead.

25 . The method of claim 1 , wherein said barcode sequence is a combinatorial assembly of a plurality of barcode segments.

26 . The method of claim 25 , wherein said plurality of barcode segments comprises at least three segments.

27 . The method of claim 1 , wherein said enzyme of (c) has terminal transferase activity.

28 . The method of claim 1 , wherein said bead is degradable upon application of a stimulus.

29 . The method of claim 28 , wherein said stimulus is a reducing agent.

30 . The method of claim 28 , wherein said droplet comprises said stimulus.

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Michael
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge
• Hardenbol Paul
• Bharadwaj Rajiv
• Zheng Xinying
• Belgrader Phillip

Classifications

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title

• C12Q2537/149

  Sequential reactions · CPC title