MULTIPLEXED SINGLE CELL GENE EXPRESSION ANALYSIS USING TEMPLATE SWITCH AND TAGMENTATION

• US 20170044525A1
• Filed: 04/28/2015
• Published: 02/16/2017
• Est. Priority Date: 04/29/2014
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: KAPER, Fiona, CANN, Gordon M., JAMSHIDI, Arash, ARAVANIS, Alex, FAN, Jian-Bing, SALATHIA, Neeraj
• Application Number:
  US15/307,329
• Publication Number:
  US 20170044525A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Presented herein are methods and compositions for multiplexed single cell gene expression analysis. Some methods and compositions include the use of droplets and/or beads bearing unique barcodes such as unique molecular barcodes (UMI).

Claims

1. A method of preparing a cDNA library from a plurality of single cells, the method comprising the steps of:

releasing mRNA from each single cell to provide a plurality of individual mRNA samples, wherein the mRNA in each individual mRNA sample is from a single cell;

synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample with a first strand synthesis primer and incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample is complementary to mRNA from a single cell, and wherein the tag comprises a cell-specific identifier sequence and optionally a unique molecular identifier (UMI) sequence;

pooling the tagged cDNA samples;

optionally amplifying the pooled cDNA samples to generate a cDNA library comprising double-stranded cDNA; and

performing a tagmentation reaction to simultaneously cleave each cDNA and incorporate an adapter into each strand of the cDNA, thereby generating a plurality of tagged cDNA fragments.

2. The method of claim 1, further comprising amplifying the tagged cDNA fragments to generate amplified tagged cDNA fragments.

3. The method of claim 2, wherein amplifying comprises adding additional sequence to the 5′

end of the amplification products.

4. The method of claim 3, wherein the additional sequence comprises primer binding sequence for amplification on a solid support.

5. The method of claim 4, further comprising amplifying the amplified tagged cDNA fragments on a solid support.

6. The method of claim 5, further comprising sequencing the amplification products on the solid support.

7. The method of claim 1, wherein the tagmentation reaction comprises contacting the double-stranded cDNA with a transposase mixture comprising adapter sequences that are not found in the first strand synthesis primer.

8. The method of claim 7, wherein the transposase mixture consists essentially of transposomes having one type of adapter sequence.

9. The method of claim 1, further comprising sequencing the tagged cDNA fragments.

10. The method of claim 9, wherein sequencing comprises 3′

tag counting.

11. The method of claim 9, wherein sequencing comprises whole transcriptome analysis.

12. The method of claim 1, first strand synthesis is performed using a mixture of random primers, the random primers further comprising a tag.

13. The method of claim 12, wherein the first strand synthesis primer comprises a double-stranded portion.

14. The method of claim 13, wherein the first strand synthesis primer reduces concatenation byproducts compared to a single-stranded first strand synthesis primer.

15. The method of claim 13, wherein the first strand synthesis primer comprises a region capable of forming a hairpin.

16. The method of claim 13, wherein the first strand synthesis primer comprises a region of RNA.

17. The method of claim 13, wherein the first strand synthesis primer is hybridized to a complementary oligonucleotide, thereby forming a double stranded portion.

18. A plurality of beads, wherein each bead comprises a plurality of oligonucleotides, each oligonucleotide comprising:

(a) a linker;

(b) an amplification primer binding site;

(c) optionally a Unique Molecular Identifier which differs for each oligonucleotide;

(d) a bead-specific sequence that is the same on each oligonucleotide on the bead but is different on other beads; and

(e) a capture sequence for capturing mRNAs and priming reverse transcription.

19. The plurality of beads of claim 18, wherein the capture sequence comprises oligo-dT.

20. The plurality of beads of claim 18, wherein each bead is in a separate droplet segregated from other beads.