Digital PCR barcoding
Methods, compositions, and kits are provided for nucleic acid analysis, including single cell analysis.
1. A method of analyzing the nucleic acid of a plurality of cells comprising:
forming a plurality of partitions, wherein each partition comprises:
a particle comprising a population of oligonucleotides having a single-stranded barcode unique for that particle and a capture sequence, wherein the oligonucleotides comprise a 5′ thiol modified uracil that is linked via a disulfide linkage to the particle and a 3′ end that is available for extension by a polymerase, and wherein the oligonucleotides comprise a hairpin that includes a uracil and wherein the particle is a hydrogel; and
a sample comprising a double-stranded target nucleic acid;
cleaving the disulfide linkage to release the oligonucleotides and to form a released 5′ end;
extending the 3′ end with a polymerase;
performing uracil excision to cleave the oligonucleotides at the hairpin thereby forming double-stranded oligonucleotides;
after the performing, ligating the 3′ end and optionally the released 5′ end of the double-stranded oligonucleotides to at least a portion of the target nucleic acid in each partition, thereby covalently attaching the double-stranded oligonucleotides to at least a portion of the target nucleic acid in each partition, wherein the ligating is performed before combining partitions;
combining the partitions; and
performing high throughput sequencing.
2. The method of claim 1 , wherein the sample comprising target nucleic acid comprises a cell containing the target nucleic acid.
3. The method of claim 2 , wherein prior to the ligating, the cell is lysed.
4. The method of claim 1 , wherein the sample comprising target nucleic acid comprises DNA.
5. The method of claim 1 , wherein the sample comprising target nucleic acid comprises long fragment DNA.
6. The method of claim 4 , wherein the DNA is double stranded.
7. The method of claim 1 , wherein the providing comprises
(a). conjugating a reverse amidite nucleotide to a plurality of precursor particles, wherein the conjugating is performed in at least four separate reactions, each reaction conjugating a different reverse amidite nucleotide, and wherein after the conjugating, the particles are combined and mixed;
(b). repeating (a) from 6-20 times, thereby generating a plurality of barcoded particles, wherein the individual barcoded particles comprise oligonucleotides comprising a plurality of copies of a particle-specific barcode, wherein the individual oligonucleotides comprise a 5′ and a 3′ end, the oligonucleotides are conjugated to the particles at the 5′ end, and the 3′ end is available for ligation and/or extension by a polymerase; and
(c). partitioning the barcoded particles, thereby generating a plurality of partition-specific barcoded partitions.
8. The method of claim 1 , wherein partitions contain a single cell or nucleic acid from a single cell.
9. The method of claim 1 , wherein the partitions are droplets surrounded by an immiscible liquid.
10. The method of claim 9 , wherein the providing comprises forming the droplets in a microfluidic device.
11. The method of claim 1 , further comprising deconvoluting the barcodes after the high throughput sequencing to associate the barcodes with cells or nucleic acids.
12. The method of claim 1 , wherein the forming comprises merging a stream of the particles with a stream of cells to form droplets comprising a cell and a particle.
13. The method of claim 12 , wherein the stream of particles comprises a cell lysis reagent that lysis cells in the droplets following the merging.
14. The method of claim 13 , wherein the cell lysis reagent is a detergent.
15. The method of claim 1 , wherein the forming comprises:
providing a mixture of droplets, wherein individual droplets comprise a cell and a particle; and
heating the droplets.
16. The method of claim 1 , wherein the extending forms a complementary sequence of the barcode.
17. The method of claim 1 , wherein the hairpin comprises the barcode.
18. The method of claim 1 , wherein the released 5′ end is ligated to the target nucleic acid.