Probe-based analysis of nucleic acids and proteins
Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.
1 . A method of analyzing a sample, comprising:
(a) providing a plurality of cells, wherein:
(i) a cell of the plurality of cells comprises a first feature, wherein the first feature is an intracellular feature; and
(ii) the cell comprises a second feature, wherein the second feature is a cell surface feature;
(b) contacting the cell with:
(i) a first feature binding group, wherein the first feature binding group couples to the first feature in the cell, wherein the first feature binding group comprises a first oligonucleotide comprising a first feature nucleic acid sequence that identifies the first feature binding group; and
(ii) a second feature binding group, wherein the second feature binding group couples to the second feature in the cell, wherein the second feature binding group comprises a second oligonucleotide comprising a second feature nucleic acid sequence that identifies the second feature binding group;
(c) after (b), partitioning the plurality of cells and a plurality of barcode molecules into a plurality of partitions, wherein a partition of the plurality of partitions comprises the cell, a first barcode molecule comprising a first barcode sequence, and a second barcode molecule comprising a second barcode sequence;
(d) after (b), generating a first barcoded nucleic acid molecule using the first feature nucleic acid sequence and the first barcode molecule, wherein the first barcoded nucleic acid molecule comprises (i) the first feature nucleic acid sequence or reverse complement thereof, and (ii) the first barcode sequence or reverse complement thereof; and
(e) after (b), generating a second barcoded nucleic acid molecule using the second feature nucleic acid sequence and the second barcode molecule, wherein the second barcoded nucleic acid molecule comprises (i) the second feature nucleic acid sequence or reverse complement thereof, and (ii) the second barcode sequence or reverse complement thereof.
2 . The method of claim 1 , further comprising, prior to or during (b), permeabilizing the cell.
3 . The method of claim 1 , further comprising contacting the cell with a fixing agent.
4 . The method of claim 3 , further comprising contacting the cell with an additional fixing agent.
5 . The method of claim 4 , wherein the fixing agent or the additional fixing agent comprises an alcohol, a ketone, or an aldehyde.
6 . The method of claim 4 , wherein the fixing agent or the additional fixing agent is selected from the group consisting of: disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS).
7 . The method of claim 4 , further comprising contacting the cell with the fixing agent and the additional fixing agent simultaneously.
8 . The method of claim 4 , further comprising contacting the cell with the fixing agent and the additional fixing agent sequentially.
9 . The method of claim 4 , wherein (b) further comprises contacting the cell with the fixing agent prior to or concurrently with contacting the cell with the first feature binding group.
10 . The method of claim 9 , wherein (b) further comprises contacting the cell with the additional fixing agent after contacting the cell with the first feature binding group.
11 . The method of claim 1 , wherein the first feature comprises a protein, a peptide, a lipid, or a carbohydrate.
12 . The method of claim 11 , wherein the first feature binding group comprises an antibody.
13 . The method of claim 1 , wherein the first feature comprises a nucleic acid molecule.
14 . The method of claim 13 , wherein the nucleic acid molecule comprises a messenger RNA (mRNA).
15 . The method of claim 13 , wherein the first feature binding group comprises a nucleic acid probe that hybridizes to a target sequence of the nucleic acid molecule.
16 . The method of claim 15 , further comprising contacting the nucleic acid molecule with an additional nucleic acid probe that hybridizes to an additional target sequence of the nucleic acid molecule.
17 . The method of claim 16 , further comprising linking the nucleic acid probe and the additional nucleic acid probe, thereby generating a linked nucleic acid molecule comprising (i) the nucleic acid probe and (ii) the additional nucleic acid probe.
18 . The method of claim 1 , wherein the plurality of partitions is a plurality of droplets or a plurality of wells.
19 . The method of claim 1 , wherein the first barcode sequence and the second barcode sequence are identical.
20 . The method of claim 1 , wherein the first barcode sequence and the second barcode sequence identify the partition from other partitions of the plurality of partitions.
21 . The method of claim 1 , wherein, in (c), the first barcode molecule or the second barcode molecule is coupled to a bead.
22 . The method of claim 1 , wherein, in (c), the first barcode molecule and the second barcode molecule are coupled to a bead, and wherein the method further comprises, prior to (d) and (e), releasing the first barcode molecule and the second barcode molecule from the bead.
23 . The method of claim 1 , wherein (d) or (e) further comprises performing a nucleic acid extension reaction.
24 . The method of claim 1 , wherein (d) or (e) further comprises performing a nucleic acid ligation reaction.
25 . The method of claim 1 , further comprising amplifying the first barcoded nucleic acid molecule or the second barcoded nucleic acid molecule.
26 . The method of claim 1 , further comprising sequencing the first barcoded nucleic acid molecule or a derivative thereof and sequencing the second barcoded nucleic acid molecule or a derivative thereof.
27 . The method of claim 1 , wherein, in (b), the cell is among the plurality of cells in a bulk sample.
28 . The method of claim 1 , wherein, in (b), the plurality of cells is dispersed across a plurality of initial partitions, and wherein the cell is in an initial partition of the plurality of initial partitions.
29 . The method of claim 28 , further comprising, after (b) and prior to (c), pooling the plurality of cells from the plurality of initial partitions.
30 . The method of claim 1 , wherein the cell further comprises a third feature, and wherein the method further comprises contacting the cell with a third feature binding group, wherein the third feature binding group couples to the third feature in the cell.