Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-12534760-B2
Application number	US-202519083328-A
Country	US
Kind code	B2
Filing date	Mar 18, 2025
Priority date	Dec 22, 2016
Publication date	Jan 27, 2026
Grant date	Jan 27, 2026

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.

First claim

What is claimed is:

1 . A composition comprising:
a partition comprising a plurality of barcode molecules, wherein the plurality of barcode molecules comprises: (a) a first barcode molecule comprising a nucleic acid barcode sequence, wherein the first barcode molecule is hybridized to a first analyte, wherein the first analyte is not messenger ribonucleic acid (mRNA) and is not hybridized to the first barcode molecule via a poly-thymine (poly-T) sequence, wherein the first analyte comprises clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleic acid (crRNA) or single guide ribonucleic acid (sgRNA); and (b) a second barcode molecule comprising the nucleic acid barcode sequence, wherein the second barcode molecule is hybridized to a second analyte, wherein the second analyte is mRNA and is hybridized to the second barcode molecule via a poly-T sequence.

2 . The composition of claim 1 , wherein barcode molecules of the plurality of barcode molecules are attached to a bead.

3 . The composition of claim 2 , wherein the bead is a gel bead.

4 . The composition of claim 2 , wherein the bead comprises polyacrylamide.

5 . The composition of claim 2 , wherein barcode molecules of the plurality of barcode molecules are releasably attached to the bead.

6 . The composition of claim 1 , wherein the partition is a partition among a plurality of partitions.

7 . The composition of claim 6 , wherein the plurality of partitions comprises at least about 1,000; 5,000; 10,000; 50,000; 100,000; 500,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000, 500,000,000; or 1,000,000,000 partitions.

8 . The composition of claim 6 , wherein the partition is a droplet, and the plurality of partitions is a plurality of droplets.

9 . The composition of claim 6 , wherein the partition is a well, and the plurality of partitions is a plurality of wells.

10 . The composition of claim 1 , wherein the first barcode molecule and the second barcode molecule further comprise a random N-mer sequence.

11 . A composition comprising:
a plurality of droplets, wherein a droplet of the plurality of droplets comprises: a gel bead having a plurality of barcode molecules attached thereto, wherein the plurality of barcode molecules comprises: (a) a first barcode molecule comprising a nucleic acid barcode sequence, wherein the first barcode molecule is hybridized to a first analyte, wherein the first analyte is not mRNA and is not hybridized to the first barcode molecule via a poly-T sequence, wherein the first analyte comprises a clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleic acid (crRNA) or a single guide ribonucleic acid (sgRNA); and (b) a second barcode molecule comprising the nucleic acid barcode sequence, wherein the second barcode molecule is hybridized to a second analyte, wherein the second analyte is mRNA and is hybridized to the second barcode molecule via a poly-T sequence, wherein the first barcode molecule and the second barcode molecule further comprise a random N-mer sequence.

12 . A composition comprising:
a partition comprising a bead having a plurality of barcode molecules attached thereto, wherein the plurality of barcode molecules comprises: (a) a first population of barcode molecules each hybridized to mRNA via poly-T sequences, wherein barcode molecules of the first population of barcode molecules each comprise a barcode sequence and a random sequence; and (b) a second population of barcode molecules each hybridized to an analyte comprising a clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleic acid (crRNA) or a single guide ribonucleic acid (sgRNA) via a sequence that is not poly-T, wherein barcode molecules of the second population of barcode molecules each comprise a barcode sequence and a random sequence; wherein barcode sequences are constant among the plurality of barcode molecules and random sequences vary among the plurality of barcode molecules.

13 . The composition of claim 12 , wherein the bead is a gel bead.

14 . The composition of claim 12 , wherein the bead comprises polyacrylamide.

15 . The composition of claim 12 , wherein the partition is a partition among a plurality of partitions.

16 . The composition of claim 15 , wherein the plurality of partitions comprises at least about 1,000; 5,000; 10,000; 50,000; 100,000; 500,000; 1,000,000: 5,000,000; 10,000,000; 50,000,000; 100,000,000, 500,000,000; or 1,000,000,000 partitions.

17 . The composition of claim 15 , wherein the partition is a droplet, and the plurality of partitions is a plurality of droplets.

18 . The composition of claim 15 , wherein the partition is a well, and the plurality of partitions is a plurality of wells.

19 . The composition of claim 11 , wherein the bead comprises polyacrylamide.

20 . The composition of claim 11 , wherein the plurality of droplets comprises at least about 1,000; 5,000; 10,000; 50,000; 100,000; 500,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000; or 1,000,000,000 droplets.

Assignees

• 10X Genomics Inc

Inventors

• Belgrader Phillip
• Bent Zachary
• Bharadwaj Rajiv
• Sreenivasa Gopalan Vijay Kumar
• Harada Josephine
• Hindson Christopher
• Rahimi Lenji Mohammad
• Lucero Michael Ybarra
• Mcdermott Geoffrey
• Meer Elliott
• Mikkelsen Tarjei Sigurd
• O'Keeffe Christopher Joachim
• Pfeiffer Katherine
• Price Andrew D
• Ryvkin Paul
• Saxonov Serge
• Stuelpnagel John R
• Terry Jessica Michele
• Wheeler Tobias Daniel
• Wu Indira
• Ziraldo Solongo Batjargal
• Boutet Stephane Claude
• Taylor Sarah
• Srinivas Niranjan

Classifications

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q1/683

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title