Single cell nucleic acid detection and analysis
Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.
1. A method of quantitatively measuring a plurality of different RNA species in a cell, comprising
isolating each single cell of a plurality of single cells in a separate reaction vessel,
releasing RNA from each isolated single cell while in the separate reaction vessel,
priming first stand synthesis of cDNA from the released RNA in each separate reaction vessel, wherein the cDNA synthesis is primed from a cDNA synthesis primer comprising (i) sample tag sequence corresponding to the single cell and (ii) a copy number barcode sequence, whereby cDNA molecules comprising the tag sequence and the barcode sequence are generated,
sequencing the cDNA molecules so as to produce DNA sequence information sufficient to identify (i) the RNA species, (ii) the tag sequences, and (iii) the copy number barcode sequence.
2. The method of claim 1 , wherein the cDNA molecules generated from the released RNA in each separate reaction vessel are pooled prior to sequencing.
3. The method of claim 1 , wherein the step of priming the synthesis of cDNA comprises contacting the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences.
4. The method of claim 3 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.
5. The method of claim 4 , wherein the copy number sequences are 6 to 30 base pairs in length.
6. The method of claim 4 , wherein the copy number barcode sequences in the set are of identical length.
7. The method of claim 4 , wherein the copy number barcode sequences are random in sequence.
8. The method of claim 4 , wherein the copy number barcode sequences in the set are separated by an error distance of at least 1.
9. The method of claim 8 , wherein the copy number barcode sequences in the set are separated by an error distance in the range of 1 to 9.
10. The method of claim 1 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule.
11. The method of claim 1 , wherein the step of priming the synthesis of cDNA comprises contacting the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences and different sample tag sequences.
12. A method of quantitatively measuring a plurality of different RNA species in a cell, comprising
physically isolating each single cell of a plurality of single cells,
releasing RNA from each isolated single cell, wherein the released RNA is physically separated from RNA released from other single cells in the plurality of single cells,
priming first stand synthesis of cDNA from each physically separated released RNA, wherein the cDNA synthesis is primed from a cDNA synthesis primer comprising (i) sample tag sequence corresponding to the single cell and (ii) a copy number barcode sequence, whereby cDNA molecules comprising the tag sequence and the barcode sequence are generated,
sequencing the cDNA molecules so as to produce DNA sequence information sufficient to identify (i) the RNA species, (ii) the tag sequences, and (iii) the copy number barcode sequence.
13. The method of claim 12 , wherein the cDNA molecules generated from the physically separated released RNA are pooled prior to sequencing.
14. The method of claim 12 , wherein the step of priming the synthesis of cDNA comprises contacting the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences.
15. The method of claim 14 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.
16. The method of claim 15 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.
17. The method claim 15 , wherein the copy number barcode sequences in the set are of identical length.
18. The method of claim 15 , wherein the copy number barcode sequences are random in sequence.
19. The method of claim 15 , wherein the copy number barcode sequences in the set are separated by an error distance of at least 1.
20. The method of claim 19 , wherein the copy number barcode sequences in the set are separated by an error distance in the range of 1 to 9.
21. The method of claim 12 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule.
22. The method of claim 12 , wherein the step of priming the synthesis of cDNA comprises contacting the released RNA with a set of cDNA synthesis primers that comprise a plurality of different copy number barcode sequences and different sample tag sequences.