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 analyzing nucleic acid sequences present in a sample, the method comprising:
(a) attaching a unique barcode sequence to substantially each of a plurality of DNA molecules in a sample to produce a plurality of differently barcoded DNA molecules, wherein each barcoded DNA molecule comprises a unique barcode sequence associated with the nucleic acid sequence of the DNA molecule;
(b) amplifying the plurality of differently barcoded DNA molecules in the sample to produce amplicons of the plurality of differently barcoded DNA molecules;
(c) sequencing each amplicon to generate sequence reads, wherein each sequence read comprises the sequence of the DNA molecule and the sequence of the associated unique barcode;
(d) sorting the sequence reads into groups, each group corresponding to a DNA molecule, wherein the sequence reads in each group comprise the same unique barcode sequence, wherein the sorting is performed by a computer implemented method;
(e) comparing two or more sequence reads within the same group.
2. The method of claim 1 , wherein step (e) comprises comparing substantially all of the sequence reads within the same group.
3. The method of claim 1 , wherein step (e) comprises determining sequence similarity between two or more sequence reads within the same group.
4. The method of claim 1 , wherein step (e) comprises aligning two or more sequence reads within the same group and determining the number of matching positions shared by the two or more sequence reads.
5. The method of claim 1 , wherein step (e) comprises comparing two or more sequence reads within the same group that can be aligned by the starting position and ending position of the sequence of the DNA molecule to the same location on a reference sequence, wherein the aligning is performed by a computer implemented method.
6. The method of claim 1 , wherein step (a) comprises attaching a unique barcode sequence to each end of substantially each of the plurality of DNA molecules in the sample.
7. The method of claim 1 , wherein the unique barcode sequences are provided in a unique barcode sequence-primer conjugate.
8. The method of claim 1 , wherein the unique barcode sequences are attached by ligating the unique barcode sequences to the DNA molecules, wherein the unique barcode sequences are present in adapters.
9. The method of claim 8 , wherein the adapters are Y-shaped adapters.
10. The method of claim 8 , wherein each adapter comprises an overhang.
11. The method of claim 8 , wherein each adapter comprises: a Y section formed by at least partially non-complementary nucleic acid sequences; a double-stranded unique barcode sequence; and an overhang.
12. The method of claim 1 , wherein the unique barcode sequences comprise pseudo-random or random sequences.
13. The method of claim 1 , wherein the unique barcode sequences are double stranded.