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 for sequencing nucleic acid molecules from a sample using barcodes for uniquely tagging molecules comprising an oligonucleotide sequence that can be used to identify an individual molecule of double-stranded cDNA in the sample, comprising:
a) applying adapters to both ends of double-stranded cDNA molecules in the sample to obtain cDNA-adapter products, wherein the adapters each comprise a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and an adapter-specific barcode on each strand of the adapter, the adapter-specific barcode being selected from a plurality of adapter-specific barcodes, and wherein each double-stranded cDNA molecule that comprises adapters has a cDNA nucleotide sequence on each strand of the double-stranded cDNA molecule;
b) amplifying both strands of the cDNA-adapter products to obtain a plurality of amplified nucleic acid products;
c) sequencing the plurality of amplified nucleic acid products, thereby obtaining a plurality of reads each comprising an adapter-specific barcode sequence corresponding to an adapter-specific barcode sequence on an adapter and a cDNA sequence corresponding to a cDNA-specific sequence on a double-stranded cDNA molecule in the sample;
d) identifying a plurality of adapter-specific barcode sequences for the plurality of reads;
e) identifying a plurality of cDNA-specific sequences for the plurality of reads; and
f) determining sequences of the double-stranded cDNA molecules in the sample using the plurality of reads obtained in (c), the plurality of adapter-specific barcode sequences identified in (d), and the plurality of cDNA-specific sequences identified in (e).
2. The method of claim 1 , wherein the plurality of adapter-specific barcodes comprises random barcode sequences.
3. The method of claim 1 , wherein the plurality of adapter-specific barcodes comprises nonrandom barcode sequences.
4. The method of claim 1 , wherein (f) comprises using reads sharing a common adapter-specific barcodes and a common cDNA-specific sequences to determine a sequence of a double-stranded cDNA molecules of the sample.
5. The method of claim 1 , wherein the adapters each comprise an adapter-specific barcode on each strand of the adapters in the double-stranded hybridized region.
6. The method of claim 5 , wherein the adapter-specific barcode is at or near an end of the double-stranded hybridized region, said end of the double-stranded hybridized region being opposite from the 3′ arm or the 5′ arm.
7. The method of claim 1 , wherein the adapters each comprise an adapter-specific barcode on each strand of the adapters in a double-stranded region of the adapters, wherein the adapter-specific barcode on one strand is complementary to the adapter-specific barcode on the other strand.
8. The method of claim 1 , wherein one or more adapter-specific barcodes and/or one or more cDNA nucleotide sequences are substantially uniquely associated with a double-stranded cDNA molecule in the sample.
9. The method of claim 1 , wherein applying adapters to both ends of double-stranded cDNA molecules comprises ligating the adapters to both ends of the double-stranded cDNA molecules.
10. A method for sequencing nucleic acid molecules from a single cell-derived sample using barcodes for uniquely tagging molecules comprising an oligonucleotide sequence that can be used to identify an individual molecule of single cell-derived double-stranded DNA in the sample, comprising:
a) applying adapters to both ends of the single cell-derived double-stranded DNA molecules in the sample to obtain DNA-adapter products, wherein the adapters each comprise a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and an adapter-specific barcode on each strand of the adapter, the adapter-specific barcode being selected from a plurality of adapter-specific barcodes, and wherein each single cell-derived double-stranded DNA molecule that comprises adapters has a nucleotide sequence on each strand of the single cell-derived double-stranded DNA molecule;
b) amplifying both strands of the DNA-adapter products to obtain a plurality of amplified nucleic acid products;
c) sequencing the plurality of amplified nucleic acid products, thereby obtaining a plurality of reads each comprising an adapter-specific barcode sequence corresponding to an adapter-specific barcode sequence on an adapter and a DNA sequence corresponding to a DNA-specific sequence on a single cell-derived double-stranded DNA molecule in the sample;
d) identifying a plurality of adapter-specific barcode sequences for the plurality of reads;
e) identifying a plurality of DNA-specific sequences for the plurality of reads; and
f) determining sequences of the single cell-derived double-stranded DNA molecules in the sample using the plurality of reads obtained in (c), the plurality of adapter-specific barcode sequences identified in (d), and the plurality of DNA-specific sequences identified in (e).
11. The method of claim 10 , wherein the plurality of adapter-specific barcodes comprises random barcode sequences.
12. The method of claim 10 , wherein the plurality of adapter-specific barcodes comprises nonrandom barcode sequences.
13. The method of claim 10 , wherein (f) comprises using reads sharing a common adapter-specific barcodes and a common DNA-specific sequences to determine a sequence of a single cell-derived double-stranded DNA molecules of the sample.
14. The method of claim 10 , wherein the adapters each comprise an adapter-specific barcode on each strand of the adapters in the double-stranded hybridized region.
15. The method of claim 14 , wherein the adapter-specific barcode is at or near an end of the double-stranded hybridized region, said end of the double-stranded hybridized region being opposite from the 3′ arm or the 5′ arm.
16. The method of claim 10 , wherein the adapters each comprise an adapter-specific barcode on each strand of the adapters in a double-stranded region of the adapters, wherein the adapter-specific barcode on one strand is complementary to the adapter-specific barcode on the other strand.
17. The method of claim 10 , wherein one or more adapter-specific barcodes and/or one or more DNA nucleotide sequences are substantially uniquely associated with a single cell-derived double-stranded single DNA molecule in the sample.
18. The method of claim 10 , wherein applying adapters to both ends of single cell-derived double-stranded DNA molecules comprises ligating the adapters to both ends of the single cell-derived double-stranded DNA molecules.