Methods for processing nucleic acid molecules

Patent metadata

Field	Value
Publication number	US-11467153-B2
Application number	US-202117393107-A
Country	US
Kind code	B2
Filing date	Aug 3, 2021
Priority date	Feb 12, 2019
Publication date	Oct 11, 2022
Grant date	Oct 11, 2022

Abstract

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

First claim

What is claimed is:

1. A method comprising:
(a) providing a partition comprising (i) a biological particle comprising (I) a tagmented fragment of a deoxyribonucleic acid (DNA) molecule and (II) a ribonucleic acid (RNA) molecule, (ii) a first nucleic acid barcode molecule comprising a first barcode sequence, (iii) a second nucleic acid barcode molecule comprising a second barcode sequence, and (iv) a splint molecule, wherein said first nucleic acid barcode molecule comprises an overhang sequence, and wherein said splint molecule comprises a first sequence complementary to a sequence of said tagmented fragment and a second sequence complementary to said overhang sequence; and
(b) within said partition: (i) hybridizing said sequence of said tagmented fragment to said splint molecule, and hybridizing said overhang sequence of said first nucleic acid barcode molecule to said splint molecule to generate a first barcoded nucleic acid product comprising (I) said first barcode sequence or a complement thereof and (II) a sequence of said DNA molecule; and (ii) using said RNA molecule and said second nucleic acid barcode molecule to generate a second barcoded nucleic acid product comprising (I) said second barcode sequence or a complement thereof and (II) a complementary DNA (cDNA) sequence of said RNA molecule.

2. The method of claim 1 , wherein said biological particle is a cell, a cell bead, or a cell nucleus.

3. The method of claim 1 , further comprising lysing or permeabilizing said biological particle within said partition to provide access to said tagmented fragment and said RNA molecule.

4. The method of claim 1 , further comprising, prior to (a), contacting an open chromatin structure of said biological particle with a transposase-nucleic acid complex to yield said biological particle comprising said tagmented fragment.

5. The method of claim 4 , wherein said transposase-nucleic acid complex comprises a first adapter and a second adapter and wherein said tagmented fragment comprises a sequence of said DNA molecule flanked by said first adapter and said second adapter.

6. The method of claim 5 , wherein (b)(i) comprises hybridizing said splint molecule to said first adapter or said second adapter of said tagmented fragment, and ligating said first nucleic acid barcode molecule and said tagmented fragment to generate said first barcoded nucleic acid product.

7. The method of claim 5 , wherein said first adapter comprises a first transposon end sequence and a first primer sequence and wherein said second adapter comprises a second transposon end sequence and a second primer sequence.

8. The method of claim 7 , wherein (b)(i) comprises:
hybridizing said splint molecule to said first primer sequence or said second primer sequence of said tagmented fragment.

9. The method of claim 8 , further comprising, prior to said ligating, using a kinase to phosphorylate said tagmented fragment or said first nucleic acid barcode molecule.

10. The method of claim 9 , wherein said kinase is a polynucleotide kinase (PNK).

11. The method of claim 7 , wherein said first primer sequence or said second primer sequence is single stranded.

12. The method of claim 1 , further comprising subjecting said first barcoded nucleic acid product to one or more nucleic acid extension reactions.

13. The method of claim 12 , wherein said one or more nucleic acid extension reactions comprise attaching one or more functional sequences to said first barcoded nucleic acid product.

14. The method of claim 13 , wherein said one or more functional sequences comprise one or more flow cell adapter sequences, one or more sequencing primer sequences, one or more sequencing primer binding sequences, or one or more sample index sequences.

15. The method of claim 1 , wherein said second nucleic acid barcode molecule comprises a capture sequence complementary to said RNA molecule and wherein (b)(ii) comprises hybridizing said capture sequence to said RNA molecule and reverse transcribing said RNA molecule to generate said second barcoded nucleic acid product.

16. The method of claim 15 , wherein said capture sequence comprises a polythymine (poly-T) sequence.

17. The method of claim 1 , wherein said partition further comprises a primer comprising a sequence complementary to said RNA molecule and wherein (b)(ii) comprises hybridizing said primer to said RNA molecule, reverse transcribing said RNA molecule to generate a cDNA molecule, performing a template switching reaction onto said second nucleic acid barcode molecule, and extending said cDNA molecule to generate said second barcoded nucleic acid product.

18. The method of claim 17 , wherein said sequence complementary to said RNA molecule comprises a polythymine (poly-T) sequence.

19. The method of claim 17 , wherein said reverse transcribing comprises use of an enzyme with terminal transferase activity that appends a polynucleotide sequence to a 3′ end of said cDNA molecule and wherein said second nucleic acid barcode molecule comprises a sequence complementary to said polynucleotide sequence to facilitate said template switching reaction.

20. The method of claim 19 , wherein said polynucleotide sequence comprises a polycytosine (polyC) sequence and wherein said sequence complementary to said polynucleotide sequence comprises a polyguanine (polyG) sequence.

21. The method of claim 20 , wherein said polyG sequence comprises a ribonucleotide.

22. The method of claim 1 , wherein said partition is a droplet.

23. The method of claim 1 , wherein said partition is a well.

24. The method of claim 1 , wherein said first barcode sequence and said second barcode sequence are a same sequence.

25. The method of claim 1 , wherein said first barcode sequence and said second barcode sequence are different.

26. The method of claim 1 , wherein said tagmented fragment or said first nucleic acid barcode molecule comprises a 5′ phosphate.

27. The method of claim 1 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are coupled to a bead.

28. The method of claim 27 , wherein said bead is a gel bead.

29. The method of claim 27 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are releasable from said bead upon application of a stimulus.

30. The method of claim 27 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are each coupled to said bead via a labile bond.

Assignees

• 10X Genomics Inc

Inventors

• Belhocine Zahra Kamila
• Bell Jason
• Sapida Jerald
• Sullivan-Bibee Katrina

Classifications

• G01N33/548

  Carbohydrates, e.g. dextran · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• G01N33/56977

  HLA or MHC typing · CPC title

• G01N33/54306

  Solid-phase reaction mechanisms · CPC title