US 10,550,429 B2Granted

Methods and systems for processing polynucleotides

Assignee10x Genomics, Inc.

InventorsJosephine Harada, Tarjei Sigurd Mikkelsen, Katherine Pfeiffer, Serge Saxonov, John R. Stuelpnagel

Life of this patent

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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.

Claims34

Dependent claims

A method of immune receptor analysis, comprising:

(a) contacting a plurality of cells with a plurality of peptide-major histocompatibility complex (MHC) molecules, wherein each peptide-MHC molecule of said plurality of peptide-MHC molecules comprises a nucleic acid reporter molecule comprising a first barcode sequence that identifies a peptide bound to an MHC molecule of said plurality of peptide-MHC molecules;
(b) partitioning cells of said plurality of cells and a plurality of nucleic acid barcode molecules comprising barcode sequences into a plurality of partitions such that a partition of said plurality of partitions comprises (i) a cell bound to a peptide-MHC molecule;
and (ii) nucleic acid barcode molecules comprising a second barcode sequence, wherein said cell comprises a messenger ribonucleic acid (mRNA) molecule encoding for an immune receptor;
(c) generating (i) a first barcoded nucleic acid molecule comprising said first barcode sequence or a complementary sequence thereof and said second barcode sequence or a complementary sequence thereof;
and (ii) a second barcoded nucleic acid molecule comprising a sequence of said mRNA molecule or a complementary sequence thereof and said second barcode sequence or a complementary sequence thereof.

2 more elements

A method of immune receptor analysis, comprising:

(a) contacting a plurality of cells with a plurality of peptide-major histocompatibility complex (MHC) multimers, wherein each MHC multimer of said plurality of peptide-MHC multimers comprises:
(i) a plurality of peptide-MHC molecules comprising a common peptide, and
(ii) a nucleic acid reporter molecule comprising (1) a first adapter sequence, (2) a reporter sequence that identifies said common peptide, and (3) a second adapter sequence;
(b) partitioning cells of said plurality of cells and a plurality of beads into a plurality of partitions, wherein said plurality of beads comprises a plurality of barcode sequences, and wherein a partition of said plurality of partitions comprises:
(i) a cell bound to an MHC multimer, wherein said cell comprises a messenger ribonucleic acid (mRNA) molecule encoding for an immune receptor;
(ii) a bead comprising a plurality of nucleic acid barcode molecules attached thereto, wherein said plurality of nucleic acid barcode molecules comprises (1) a common barcode sequence and (2) a capture sequence configured to perform a template switching reaction;
and
(iii) a primer comprising a poly-T sequence;
(c) in said partition, hybridizing said first adapter sequence of said reporter molecule to said capture sequence and performing a nucleic acid extension reaction to generate a first barcoded nucleic acid molecule comprising (i) said common barcode sequence or a complementary sequence thereof, (ii) said reporter sequence or a complementary sequence thereof, and (iii) said second adapter sequence or a complementary sequence thereof;
(d) in said partition, (i) hybridizing a poly-A sequence of said mRNA molecule to said poly-T sequence of said primer, (ii) performing a reverse transcription reaction to generate a complementary deoxyribonucleic acid (cDNA) molecule, and (iii) performing a template switching reaction onto a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to generate a second barcoded nucleic acid molecule comprising (1) said common barcode sequence or a complementary sequence thereof, and (2) a sequence of said mRNA molecule or a complementary sequence thereof.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/426,762Filed 30 May 2019 · continuation in part
Priority: 22 Dec 2016
Published: US 2019/0338353 A17 Nov 2019
Granted: 4 Feb 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

C12Q 1/6853
Nucleic acid amplification reactions: using modified primers or templates
C12Q 1/6874
Methods for sequencing: involving nucleic acid arrays, e.g. sequencing by hybridisation
C12N 15/1075
Isolating an individual clone by screening libraries: by coupling phenotype to genotype, not provided for in other groups of this subclass
C12Q 1/6804
Involving nucleic acids: nucleic acid analysis using immunogens

Family

Application62/438,341Us provisional application · filed 22 Dec 2016
Application15/720,085Parent of this continuation · filed 29 Sep 2017
PCT/US2017/068320Parent of this continuation · filed 22 Dec 2017
Application15/933,299Parent of this continuation-in-part · filed 22 Mar 2018
US 2019/0338353 A1Pre-grant publication · published 7 Nov 2019
US 10,011,872 B1Parent of this continuation
Application15/933,299Parent of this continuation
US 10,480,029 B2Related patent
US 2018/0179590 A1Published application
US 2018/0282803 A1Published application
US 10,550,429 B2This patent

From the US filing history. Foreign family members aren’t listed.