Methods of single-cell polypeptide sequencing

Patent metadata

Field	Value
Publication number	US-2021139973-A1
Application number	US-202017082918-A
Country	US
Kind code	A1
Filing date	Oct 28, 2020
Priority date	Oct 28, 2019
Publication date	May 13, 2021
Grant date	—

Abstract

Provided herein are methods of single-cell polypeptide and/or polynucleic acid sequencing, which facilitate the direct sequencing of a single cell without amplification. Also provided herein are compositions, kits and devices useful for the same.

First claim

1 . A method comprising directly sequencing, in parallel, the proteome of a single cell and optionally sequencing the genome and/or transcriptome of the single cell, and/or optionally detecting one or more metabolite of the single cell.

2 . The method of claim 1 , wherein the method comprises:
(i) providing a cell sample comprising the composition of a single cell; (ii) contacting the cell sample with a barcode component to produce a labeled sample comprising barcoded molecules, wherein the barcoded molecules comprise barcoded polypeptides; and (iii) sequencing the polypeptides of the labeled sample.

3 . The method of claim 1 , wherein the method comprises:
(i) providing a cell sample comprising the composition of a single cell; (ii) contacting the cell sample with a barcode component to produce a labeled sample comprising barcoded molecules, wherein the barcoded molecules comprise barcoded polynucleic acids; and (iii) sequencing the polynucleic acids of the labeled sample, optionally wherein the sequencing comprises long-read sequencing applications, short-read sequencing applications, or hybrid assembly applications.

4 .- 9 . (canceled)

10 . The method of claim 2 , wherein the barcoded molecules of the labeled sample further comprise barcoded DNA, barcoded RNA, barcoded cDNA, and/or barcoded metabolites, optionally wherein the method further comprises amplifying the barcoded DNA, barcoded RNA, and/or the barcoded cDNA.

11 . (canceled)

12 . The method of claim 2 , wherein (ii) comprises:
(a) contacting the cell sample with a first barcode component to produce a first sample comprising barcoded polypeptides; (b) isolating the barcoded polypeptides from the first sample, thereby generating a second sample comprising the barcoded polypeptides and a third sample comprising the genome, transcriptome, and/or metabolome of the single cell in the cell sample; and (c) contacting the third sample with an additional barcode component, to produce a fourth sample comprising barcoded DNA, barcoded RNA, barcoded cDNA, and/or barcoded metabolites; wherein the labeled sample comprises the second sample and the fourth sample;
optionally wherein the third sample in (b) comprises:
(i) a first subsample comprising the genome and transcriptome of the single cell and a second subsample comprising the metabolome of the single cell;
(ii) a first subsample comprising the genome and metabolome of the single cell and a second subsample comprising the transcriptome of the single cell;
(iii) a first subsample comprising the metabolome and transcriptome of the single cell and a second subsample comprising the genome of the single cell; or
(iv) a first subsample comprising the genome of the single cell, a second subsample comprising the transcriptome of the single cell, and a third subsample comprising the metabolome of the single cell.

13 .- 15 . (canceled)

16 . The method of claim 2 , wherein (ii) comprises:
(a) contacting the cell sample with a first barcode component to produce a first sample comprising barcoded DNA, barcoded RNA, and/or barcoded cDNA; (b) amplifying the barcoded DNA, barcoded RNA, and/or the barcoded cDNA of the first sample; and (c) contacting the first sample with a second barcode component, to produce a second sample comprising barcoded polypeptides and/or barcoded metabolites; wherein the labeled sample comprises the second sample.

17 . The method of claim 2 , wherein (ii) comprises:
(a) contacting the cell sample with a first barcode component to produce a first sample comprising barcoded DNA, barcoded RNA, and/or barcoded cDNA; (b) isolating the barcoded DNA, barcoded RNA, and/or barcoded cDNA from the first sample, thereby generating a second sample comprising the barcoded DNA and/or barcoded cDNA and a third sample comprising the proteome and/or metabolome of the single cell in the cell sample; and (c) contacting the third sample with an additional barcode component, to produce a fourth sample comprising barcoded polypeptides and/or the barcoded metabolites; wherein the labeled sample comprises the second sample and the fourth sample optionally wherein: the third sample in (b) comprises a first subsample comprising the proteome of the single cell and a second subsample comprising the metabolome of the single cell; the additional barcode component in (c) comprises a second or third barcode component; or a combination thereof.

18 .- 20 . (canceled)

21 . The method of claim 10 , further comprising sequencing the barcoded polypeptides, the barcoded DNA, barcoded RNA, and/or barcoded cDNA of the labeled sample.

22 . The method of claim 10 , further comprising detecting and optionally quantifying one or more of the barcoded metabolites of the labeled sample.

23 .- 24 . (canceled)

25 . The method of claim 21 , wherein the sequencing comprises:
(a) detecting the barcode identities of the barcoded molecules of the labeled sample, thereby determining the origins of the barcoded molecules; and (b) sequencing, in parallel, the barcoded polypeptides in the labeled sample, thereby determining at least the partial amino acid sequences of the barcoded polypeptides;
wherein (a) occurs before, after, or concurrently with (b).

26 .- 35 . (canceled)

36 . The method of claim 1 , wherein the sequencing comprises:
(a) contacting a single polypeptide molecule with one or more terminal amino acid recognition molecules; and (b) detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with successive amino acids exposed at a terminus of the single polypeptide while the single polypeptide is being degraded, thereby sequencing the single polypeptide molecule.

37 . The method of claim 1 , wherein the sequencing comprises:
(a) contacting a single polypeptide molecule with a composition comprising one or more terminal amino acid recognition molecules and a cleaving reagent; and (b) detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with a terminus of the single polypeptide molecule in the presence of the cleaving reagent, wherein the series of signal pulses is indicative of a series of amino acids exposed at the terminus over time as a result of terminal amino acid cleavage by the cleaving reagent.

38 . The method of claim 1 , wherein the sequencing comprises:
(a) identifying a first amino acid at a terminus of a single polypeptide molecule; (b) removing the first amino acid to expose a second amino acid at the terminus of the single polypeptide molecule, and (c) identifying the second amino acid at the terminus of the single polypeptide molecule, wherein (a)-(c) are performed in a single reaction mixture.

39 . The method of claim 1 , wherein the sequencing comprises:
(a) contacting a single polypeptide molecule with one or more amino acid recognition molecules that bind to the single polypeptide molecule; (b) detecting a series of signal pulses indicative of association of the one or more amino acid recognition molecules with the single polypeptide molecule under polypeptide degradation conditions; and (c) identifying a first type of amino acid in the single polypeptide molecule based on a first characteristic pattern in the series of signal pulses.

40 . The method of claim 1 , wherein the sequencing comprises:
(a) obtaining data during a polypeptide degradation process; (b) analyzing the data t

Assignees

• Quantum Si Inc

Inventors

• Dyer Matthew
• Reed Brian

Classifications

• G01N33/6842

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• G01N33/6818 Primary

  Sequencing of polypeptides · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2563/185

  Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals · CPC title