Multiplex analysis of single cell constituents

Patent metadata

Field	Value
Publication number	US-11092607-B2
Application number	US-201615771987-A
Country	US
Kind code	B2
Filing date	Oct 27, 2016
Priority date	Oct 28, 2015
Publication date	Aug 17, 2021
Grant date	Aug 17, 2021

Abstract

The present invention relates to methods for high multiplex protein or cellular constituent analysis in single cells or single isolated aggregations of cellular constituents. The methods provide for embedding cells or isolated aggregations of cellular constituents in a hydrogel mesh and labeling of cellular constituents with labeling ligands linked to a nucleic acid tag. Cellular constituents can be determined using sequencing methods.

First claim

What is claimed is:

1. A method of assaying single cells or aggregations of cellular constituents from single cells, comprising:
(a) segregating a plurality of single cells or single aggregations of cellular constituents, each from a single cell, into separate reaction vessels comprising monomers of a polymerizable gel and a fixing agent;
(b) polymerizing the gel to embed the plurality of single cells or single aggregations of cellular constituents from single cells in discrete polymer matrices, whereby the cellular constituents of the single cells or aggregations are embedded in the discrete polymer matrices;
(c) pooling the discrete polymer matrices into a single reaction vessel;
(d) incubating the cellular constituents embedded in the pooled polymer matrices with one or more labeling ligands with specific binding affinity for one or more target cellular constituents to produce one or more labeled cellular constituents in the polymer matrices,
wherein each of the one or more labeling ligands comprise a bound oligonucleotide label comprising a unique constituent identifier (UCI) sequence specific for identifying the labeling ligand, wherein the UCI comprises 4 to 30 nucleotides,
optionally, wherein the oligonucleotide label further comprises a unique molecular identifier (UMI) sequence comprising 4-20 nucleotides,
wherein the cellular constituents comprise a protein, RNA, and/or a DNA molecule,
and wherein the incubation comprises binding conditions under which the one or more labeling ligands will bind to the cellular constituents within the polymer matrices, and the incubation further comprises washing the polymer matrices to remove unbound labeling ligands from the polymer matrices; and
(e) sequencing the oligonucleotide labels for single polymer matrices, optionally amplifying the oligonucleotide labels before sequencing, whereby detecting the UCI by sequencing indicates the presence of the target cellular constituent in the segregated aggregations of cellular constituents.

2. The method of claim 1 , further comprising segregating the discrete polymer matrices comprising the labeled constituents after incubating in step (d) and before sequencing in step (e),
wherein segregating the discrete polymer matrices comprises sorting single discrete matrices into separate reaction vessels; or
wherein segregating the discrete polymer matrices comprises forming discrete unique-identifier-transfer compositions, each comprising the cellular constituents embedded in a discrete polymer matrix and a transfer particle, wherein:
(a) the oligonucleotide label further comprises a capture sequence, and the UCI and capture sequence are together releasably attached to the labeling ligand;
(b) the labelling ligand is bound to the target cellular constituent; and,
(c) the transfer particle comprises:
(i) a capture-binding-sequence having specific binding affinity for the capture sequence attached to the UCI, and,
(ii) a unique source identifier (USI) sequence that is unique to each transfer particle; and

wherein the method further comprises releasing the UCI from the labeled ligand, under conditions within the unique-identifier-transfer composition so that the released capture sequence binds to the capture-binding-sequence on the transfer particle, thereby transferring the UCI to the transfer particle.

3. The method of claim 2 , wherein segregating the discrete polymer matrices into separate reaction vessels further comprises generating a USI for each discrete polymer matrix by a split pool method,
wherein the oligonucleotide label further comprises a universal ligation handle (ULH) sequence capable of hybridization to a complementary sequence on a first index nucleotide sequence,
optionally, wherein the ligation handle comprises a restriction site for producing a sequence complementary with a first index sequence, and wherein the method further comprises digestion with a restriction enzyme,
optionally, wherein the ligation handle comprises a nucleotide sequence complementary with a ligation primer sequence and wherein the sequence complementary with a first index sequence is produced by hybridization of the ligation primer to the ligation handle,
wherein the first index nucleotide sequence comprises a sequence complementary to a final index sequence or a middle index sequence,
wherein the middle index sequence comprises a sequence complementary to the first index sequence and to the final index sequence or optionally to another middle index sequence and final index sequence,
wherein the final index sequence has a sequence complementary to the preceding index sequence,
wherein the first, middle, and final index sequences are selected from a plurality of unique sequences comprising compatible DNA sequences for hybridization and 10 to 30 base pairs of unique sequence,
optionally, wherein either the first, middle, or final index sequence further comprises a unique molecular identifier (UMI) sequence comprising 4-20 nucleotides, and
wherein the split pool method comprises:
(a) splitting the pool of discrete polymer matrices into separate pools of polymer matrices, each containing a unique first index sequence;
(b) ligating the first index sequence to the ligation handle or hybridizing and extending the first index sequence on the ligation handle;
(c) pooling the discrete polymer matrices;
(d) optionally,
(i) splitting the pool of discrete polymer matrices into separate pools each containing a unique middle index sequence;
(ii) ligating the middle index sequence to the first index sequence or hybridizing and extending the middle index sequence on the first index; and
(iii) pooling the discrete polymer matrices;

(e) optionally, repeating step (d) with another middle index sequence;
(f) splitting the pool of discrete polymer matrices into pools containing a unique final index sequence; and
(g) ligating the final index sequence to the preceding index sequence or hybridizing and extending the final index sequence on the preceding index sequence index,

whereby each discrete polymer matrix comprises a USI comprising a combination of indexes.

4. The method of claim 1 , wherein the aggregation of cellular constituents is an extracellular vesicle, an organelle, or an organized subcomponent thereof.

5. The method of claim 1 , wherein the steps of (a) segregating and (b) polymerizing is carried out in an aqueous aliquot or in a droplet formed by an aqueous solution in oil emulsion, and/or
wherein the polymer matrix is a hydrogel, and/or
wherein the method is a multiplex assay with a plurality of labeling ligands, each labeling ligand having a distinct UCI, and/or
wherein the labeling ligand is non-covalently bound to the target cellular constituent, and/or
wherein the method further comprises treating the cellular constituents embedded in the polymer matrices with a detergent so as to remove lipids from the polymer matrices before incubating the cellular constituents with the labeling ligand, and/or
wherein the method further comprises quantitating the relative differences in the amount of a cellular constituent between aggregations of cellular constituents.

6. The method of claim 2 , further comprising pooling the oligonucleotide labels comprising a USI from the plurality of polymer matrices and sequencing the pooled UCI sequences and USI sequences, or
further comprising pooling the oligonucleotide labels comprising a USI and UMI from a plurality of polymer matrices and sequencing the pooled UCI sequences, USI sequences, and UMI sequences.

7. The method of claim 1 , wherein the labeling ligand is an antibody or an antibody fragment, an aptamer, or an oligonucleotide sequence configured to hybridize to a transcript specific region or guide RNA sequence.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• The Board Inst Inc

Inventors

• Gaublomme Jellert
• Regev Aviv

Classifications

• C12Q1/6823

  Release of bound markers · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title