Methods of identifying nucleic acids from a single cell using an electrical cell-trapping array

• US 10,640,824 B1
• Filed: 05/30/2017
• Issued: 05/05/2020
• Est. Priority Date: 06/03/2016
• Status: Active Grant

• Current Assignee: Verily Life Sciences LLC (Alphabet Inc.)
• Original Assignee: Verily Life Sciences LLC (Alphabet Inc.)
• Inventors: Peikon, Ian, Homyk, Andrew
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/608,364
• Time in Patent Office: 1,071 Days
• Field of Search: 506 3
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

Provided herein are methods, systems, kits and compositions for identifying nucleic acids of interest from individual cells. The methods include isolating single cells on electrodes and identifying nucleic acids of interest from the isolated cells by sequencing.

Claims

1. A method of identifying nucleic acids of interest within single cells, comprising:

contacting a solid surface with a plurality of cells wherein the solid surface comprises an array of identical features, wherein each identical feature comprises a library of distinct oligonucleotides, wherein each oligonucleotide comprises at least one unique molecular identifier sequence and a sequence complimentary to one of a plurality of nucleic acids of interest, wherein the surface further comprises a planar electrode of negative charge and wherein the planar electrode is positioned over the solid surface with space between the solid surface and planar electrode of negative charge;

immobilizing at least one of the cells at one of the identical features on the array on the solid surface by application of an electric field;

releasing the nucleic acids of interest from the cells;

hybridizing the oligonucleotides to the nucleic acids of interest;

releasing the hybridized nucleic acids of interest and oligonucleotides from the solid surface;

sequencing the released hybridized nucleic acids of interest and oligonucleotides to generate sequence data; and

analyzing the sequence data to identify nucleic acids of interest within the single cells.

2. The method of claim 1, wherein analyzing comprises sorting the read lengths of the data obtained from the sequencing step and identifying sequences of nucleic acids of interest present in read lengths exceeding a predetermined threshold.

3. The method of claim 1, wherein analyzing comprises quantifying the nucleic acids of interest.

4. The method of claim 1, wherein each oligonucleotide further comprises:

a sequencing adaptor sequence; and

a barcode sequence corresponding to the location of the identical feature on the array on the solid surface.

5. The method of claim 1, wherein the array comprises at least 10, 100, 1,000, 10,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,00, 700,000, 800,000, 900,000, 1,000,000, or 10,000,000 identical features.

6. The method of claim 1, wherein analyzing the sequence data comprises assigning the nucleic acids of interest to a single cell.

7. The method of claim 6, wherein assigning the nucleic acid of interest to a single cell comprises identifying in the sequence data the presence of the common sequence corresponding to the location of identical feature on the array on the solid surface to which the cell was localized.

8. The method of claim 1, wherein the solid surface comprises a plurality of electrodes, wherein each electrode is individually associated with an identical feature on the array.

9. The method of claim 8, wherein each electrode has a diameter of less than 2 microns.

10. The method of claim 8, wherein each electrode has a diameter of less than 100 microns.

11. The method of claim 8, wherein each electrode binds to a single cell.

12. The method of claim 8, wherein the unique molecular identifier sequence is between 5 and 30 nucleotides in length.

13. The method of claim 1, wherein the cells on the solid support surface are visualized by microscopy.

14. The method of claim 13, wherein the visualization is used to guide movement of cells to electrodes.

15. The method of claim 13, wherein image data produced from the microscopy is matched to sequencing data obtained from corresponding cells.

16. The method of claim 1, wherein the cells are moved to the distinct locations on the solid support surface by electrophoresis.

17. The method of claim 1, wherein the identical feature is configured to contain a single cell.

18. A solid surface comprising a plurality of electrodes and an array of identical features, wherein each identical feature comprises a library of distinct oligonucleotides wherein each oligonucleotide comprises:

i) a sequencing adaptor sequence;

ii) a barcode sequence corresponding to the location of the identical feature on the array on the solid surface;

iii) a unique molecular identifier sequence; and

iv) a sequence complimentary to one of a plurality of nucleic acids of interest

wherein the surface further comprises a planar electrode of negative charge and wherein the planar electrode is positioned over the solid surface with space between the solid surface and planar electrode of negative charge.

19. The solid support surface of claim 18, wherein each electrode in the plurality of electrodes is located at an identical feature on the array and the library of distinct oligonucleotides is bound to the electrode.

20. The solid support surface of claim 18, wherein each electrode has a diameter less than 2 microns.

21. The solid surface of claim 18, wherein each identical feature is configured to contain a single cell.

22. A kit comprising the solid surface of claim 18 and instructions for use.