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 composition comprising:
a plurality of tagged cDNA molecules each labeled with (i) a copy number barcode sequence and (ii) a sample barcode sequence that identifies an isolated single cell providing RNA from which the cDNA was generated, wherein the plurality of tagged cDNA molecules are derived from a plurality of isolated single cells, wherein the sample barcode sequence identifies the isolated single cell providing RNA from which the tagged cDNA molecules were generated.
2. The composition of claim 1 , wherein the tagged cDNA molecules are in a library prepared for sequencing in a high throughput DNA sequencer.
3. The composition of claim 1 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.
4. The composition of claim 3 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.
5. The composition of claim 1 , wherein the copy number barcode sequences are of identical length.
6. The composition of claim 1 , wherein the copy number barcode sequences are random in sequence.
7. The composition of claim 1 , wherein the copy number barcode sequences form a set of copy number barcode sequences that are separated by an error distance of at least 1.
8. The composition of claim 7 , wherein copy number barcode sequences are separated by an error distance in the range of 1 to 9.
9. A composition of tagged cDNA molecules formed by a method comprising the steps:
physically isolating a plurality of single cells to produce isolated single cells,
releasing RNA from the isolated single cells, wherein the released RNA is physically separated from RNA released from other single cells,
forming tagged cDNA from the released RNA, wherein the tagged cDNA comprises a (i) sample barcode sequence corresponding to the single cell and (ii) a copy number barcode sequence, and
pooling the tagged cDNA derived from the different isolated single cells.
10. The composition of claim 9 , further comprising the step of modifying the pooled tagged cDNA so as to prepare the pooled tagged cDNA for sequencing in a high throughput DNA sequencer.
11. The composition of claim 9 , wherein the copy number barcode sequences are 3 to 75 base pairs in length.
12. The composition of claim 11 , wherein the copy number barcode sequences are 6 to 30 base pairs in length.
13. The composition of claim 9 , wherein the copy number barcode sequences are of identical length.
14. The composition of claim 9 , wherein the copy number barcode sequences are random in sequence.
15. The composition of claim 9 , wherein the copy number barcode sequences form a set of copy number barcode sequences that are separated by an error distance of at least 1.
16. The composition of claim 15 , wherein the copy number barcode sequences are separated by an error distance in the range of 1 to 9.
17. The composition of claim 9 , wherein the forming of the 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 barcode sequences, wherein the contacting occurs when the RNA is physically separated from RNA released from other single cells.
18. The composition of claim 17 , wherein the cDNA synthesis primers can hybridize to a polyA tail of an mRNA molecule.
19. The composition of claim 1 , wherein, the copy number barcode sequences form a set of copy number barcode sequences that are common to tagged cDNA molecules derived from the plurality of isolated single cells.