HIGH-THROUGHPUT SINGLE-CELL ANALYSIS COMBINING PROTEOMIC AND GENOMIC INFORMATION

• US 20160244828A1
• Filed: 02/17/2016
• Published: 08/25/2016
• Est. Priority Date: 02/19/2015
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Mason, Clark
• Granted Patent: US 10,697,010 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/626 being a flow cytometer
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1402 Data analysis by thresholding or gating operations performed on the acquired signals or stored data

Abstract

Disclosed herein are methods for single-cell sequencing. In some examples, the methods include enriching a sample comprising a plurality of cells for cells of interest to produce an enriched cell sample; isolating one or more cells of interest in the enriched cell sample; and obtaining sequence information of one or more polynucleotides from each of the one or more isolated cells. Obtaining sequence information may include generating a molecularly indexed polynucleotide library from the one or more isolated cells. Enriching the sample may include focusing cells of interest in the sample using acoustic focusing.

Claims

1. A method of single-cell polynucleotide sequencing, the method comprising:

enriching a sample comprising a plurality of cells for cells of interest to produce an enriched cell sample;

isolating one or more cells of interest from the enriched cell sample; and

obtaining sequence information of one or more polynucleotides from each of the one or more isolated cells, wherein obtaining sequence information comprises generating a molecularly indexed polynucleotide library from the one or more isolated cells.

2. The method of claim 1, wherein enriching the sample comprises focusing the cells of interest in the sample.

3. The method of claim 2, wherein focusing the cells of interest in the sample comprises focusing the cells of interest in the sample into a core stream of cells.

4. The method of claim 2, wherein focusing the cells of interest in the sample is based on cell size.

5. The method of claim 2, wherein focusing the cells of interest in the sample comprises acoustic focusing.

6. The method of claim 5, wherein acoustic focusing comprises:

suspending the plurality of cells in the sample in an elongated fluid-filled channel; and

exposing said channel to an axial acoustic standing wave field parallel to the direction of flow, wherein said axial acoustic standing wave field drives the plurality of cells to positions of potential force minimum along the center axis of said channel to result in uniformly spaced cells.

7. The method of claim 1, wherein isolating the one or more cells of interest in the sample comprises sorting cells in the enriched cell sample with a flow cytometer.

8. The method of claim 7, wherein isolating the one or more cells of interest from the enriched cell sample further comprises depositing the one or more cells of interest in the enriched cell sample into one or more microtiter plates.

9. The method of claim 1, wherein the one or more polynucleotides comprise ribonucleic acids (RNAs).

10. The method of claim 9, wherein the RNAs are messenger RNAs (mRNAs).

11. The method of claim 10, wherein the sequence information comprises transcript counts of at least 10 genes.

12. The method of claim 1, wherein no more than 500 cells are isolated from the enriched cell sample.

13. The method of claim 1, wherein obtaining sequence information further comprises sequencing the molecularly indexed polynucleotide library.

14. The method of claim 13, wherein sequencing the molecularly indexed polynucleotide library comprises deconvoluting the sequencing result from sequencing the library.

15. The method of claim 1, wherein the sample comprises a biological sample, a clinical sample, an environmental sample, or a biological fluid, tissue or cell from a patient.

16. The method of claim 1, wherein the cells of interest in the sample comprise stem cells, cancer cells, blood cells, peripheral blood mononuclear cells, circulating tumor cells, breast cancer cells, cells at a cell cycle phase of desire, or a combination thereof.

17. The method of claim 15, further comprising determining genotype, phenotype, or one or more genetic mutation of the patient based on the obtained sequence information.

18. The method of claim 15, further comprising predicting susceptibility of the patient to one or more diseases.

19. The method of claim 18, wherein at least one of the one or more diseases is cancer or a hereditary disease.

20. The method of claim 1, wherein less than 10% of total number of cells in the sample are the cells of interest in the sample.