Droplet-based method and apparatus for composite single-cell nucleic acid analysis
The present invention generally relates to a combination of molecular barcoding and emulsion-based microfluidics to isolate, lyse, barcode, and prepare nucleic acids from individual cells in a high-throughput manner.
1. A plurality of at least a thousand RNA capture microbeads wherein each RNA capture microbead comprises a plurality of capture oligonucleotides attached to the microbead surface, each capture oligonucleotide comprising:
i. a linker;
ii. a sequence for use as a sequencing priming site, wherein the sequence is identical on all beads;
iii. a cell-of-origin barcode sequence that is the same for all capture oligonucleotides on the same bead but differs from the barcode sequence of capture oligonucleotides on other beads, wherein the maximum complexity of cell-of-origin barcodes for the plurality of RNA capture microbeads is 4 n where n is the length of the cell-of-origin barcode sequence and n is at least 6;
iv. a unique molecular identifier (UMI) sequence that is different for each capture oligonucleotide on the same bead;
v.a capture sequence that binds to cellular RNA and primes reverse transcription; and
vi. optionally at least one additional oligonucleotide sequence which provides substrates for downstream molecular-biological reactions,
and wherein the cell-of-origin barcode sequence is contiguous with the UMI sequence.
2. The plurality of RNA capture microbeads of claim 1 , wherein the cell-of-origin barcode ranges from 6 to 1000 nucleotides in length.
3. The plurality of RNA capture microbeads according to claim 1 , wherein the capture sequence that binds to cellular RNA and primes reverse transcription is an oligo dT sequence.
4. The plurality of RNA capture microbeads according to claim 1 , wherein the linker is a non-cleavable, straight-chain polymer.
5. The plurality of RNA capture microbeads according to claim 1 , wherein the linker is a chemically-cleavable, straight-chain polymer.
6. The plurality of RNA capture microbeads according to claim 1 , wherein the linker is a non-cleavable optionally substituted hydrocarbon polymer.
7. The plurality of RNA capture microbeads according to claim 1 , wherein the linker is a photolabile optionally substituted hydrocarbon polymer.
8. The plurality of RNA capture microbeads according to claim 1 , wherein the linker is a polyethylene glycol.
9. The plurality of RNA capture microbeads to claim 1 , wherein the linker is a PEG- 3 to PEG- 24 .
10. The plurality of RNA capture microbeads of claim 1 , which comprises the at least one additional oligonucleotide sequence which provides substrates for downstream molecular-biological reactions.
11. The plurality of RNA capture microbeads of claim 10 , wherein the downstream molecular-biological reactions are for reverse transcription of mature mRNAs; capturing specific portions of the transcriptome, priming for DNA polymerases and/or similar enzymes; or priming throughout the transcriptome or genome.
12. The plurality of RNA capture microbeads of claim 10 , wherein the additional oligonucleotide sequence comprises an oligo-dT sequence.
13. The plurality of RNA capture microbeads of claim 10 , wherein the additional oligonucleotide sequence comprises a primer sequence which comprises the same sequence across all beads in the plurality of beads.
14. The plurality of RNA capture microbeads of claim 10 , wherein the additional oligonucleotide sequence comprises an oligo-dT sequence and a primer sequence.
15. The plurality of RNA capture microbeads of claim 1 , wherein the capture oligonucleotide comprises at least one chemically modified nucleotide.
16. The plurality of RNA capture microbeads of claim 1 , wherein the bead material is porous.
17. The plurality of RNA capture microbeads of claim 16 , wherein the bead material is methacrylate resin.
18. The plurality of RNA capture microbeads of claim 1 , wherein the plurality of RNA capture microbeads comprise 100,000 to 10 million uniquely barcoded RNA capture microbeads.
19. The plurality of RNA capture microbeads of claim 1 , wherein the cell-of-origin barcode is 6 to 12 nucleotides in length.