METHODS AND COMPOSITIONS FOR NUCLEIC ACID ANALYSIS
Provided herein are methods, compositions, and kits for assays, many of which involve amplification reactions such as digital PCR or droplet digital PCR. The assays may be used for such applications as sequencing, copy number variation analysis, and others. In some cases, the assays involve subdividing a sample into multiple partitions (e.g., droplets) and merging the partitions with other partitions that comprise adaptors with barcodes.
1 .- 45 . (canceled)
46 . A method of analyzing cells, the method comprising, separating individual cells into unique partitions comprising uniquely-barcoded adaptor oligonucleotides;
attaching the uniquely-barcodes adaptor oligonucleotides to polynucleotides from the cells to form barcoded polynucleotides;
pooling the barcoded polynucleotides from partitions; and
sequencing the barcoded polynucleotides.
47 . the method of claim 46 , wherein the partitions are droplets.
48 . The method of claim 46 , wherein the polynucleotides from the cells are reverse transcribed mRNA (cDNA).
49 . The method of claim 48 , further comprising reverse transcribing mRNA from the cells in the partitions with partition-specific barcoded primers to form barcoded cDNAs.
50 . The method of claim 49 , wherein the partition is an outer droplet and a reverse transcriptase is sequestered in an inner droplet in the outer droplet, and wherein the inner droplet is burst to allow the transcriptase to contact the mRNA.
51 . The method of claim 50 , wherein the inner droplet is burst by heating the outer and inner droplet.
52 . The method of claim 48 , further comprising incorporating unique barcodes into the cDNAs following the reverse transcribing.
53 . The method of claim 46 , wherein the polynucleotides from the cells are genomic DNA.
54 . The method of claim 53 , further comprising determining copy number variation (CNV) from the sequencing.
55 . The method of claim 46 , further comprising amplifying the genomic DNA before and/or after adding adaptors with barcodes.
56 . The method of claim 47 , wherein multiple-displacement amplification (MDA) is performed within a droplet on a cell's genome, prior to fragmentation and adaptor ligation.
57 . The method of claim 53 , wherein the partitions comprise methyl-sensitive endonucleases.
58 . The method of claim 57 , wherein the methyl-sensitive endonucleases digest methylated sites.
59 . The method of claim 57 , comprising merging partitions comprising uniquely barcoded adaptors with partitions comprising sample polynucleotides.
60 . The method of claim 59 , wherein the uniquely barcoded adaptors are ligated to polynucleotides in the partition before or after digestion with the methyl-sensitive enzyme.
61 . The method of claim, wherein the genomic DNA is treated with bisulfite, converting unmethylated cytosines to uracil.
62 . The method of claim 46 , wherein the attaching comprises ligating the uniquely-barcodes adaptor oligonucleotides to the polynucleotides from the cells.
63 . The method of claim 46 , wherein the attaching comprises amplifying the polynucleotides from the cells with the uniquely-barcodes adaptor oligonucleotides.