Methods and compositions for single cell genomics
View Patent ↗Presented are methods and compositions for obtaining sequence information from one or more individual cells. The methods are useful for obtaining sequence information for a single nucleotide sequence, and for multiplex generation of sequence information from one or more individual cells.
1. A method of providing sequence information regarding at least one target nucleic acid sequence in a plurality of nucleic acid molecules from an individual cell comprising:
(a) amplifying a plurality of nucleic acid molecules in situ within a cell, thereby generating a set of amplified nucleic acid molecules, wherein the set of amplified nucleic acid molecules comprise the at least one target nucleic acid sequence;
(b) for each amplified nucleic acid sequence of said set of amplified nucleic acid molecules, annealing in situ a padlock probe that is designed to anneal to the at least one target nucleic acid sequence, wherein the padlock probe comprises two sequence-specific capturing arms on the 5′ and 3′ end of the probe, wherein the capturing arms are ligatable when each are annealed to the target nucleic acid sequence, wherein the padlock probe further comprises at least a first decoder sequence and a second decoder sequence that do not anneal to said amplified nucleic acid molecules, and wherein the padlock probe can be identified based upon generating a code from decoding the at least first decoder sequence and the second decoder sequence by using sequential hybridization with fluorescently labeled decoder probes, and identifying the padlock probe by matching said code with a reference code that is assigned for each padlock probe, wherein each padlock probe that hybridizes to a different target nucleic acid sequence has a different reference code;
(c) hybridizing a first fluorescently labeled decoder probe to said first decoder sequence, wherein the first fluorescently labeled decoder probe that is not hybridized to said first decoder sequence is removed by washing;
(d) detecting a plurality of fluorescent signals indicative of said hybridizing of the first fluorescently labeled decoder probe to said first decoder sequence;
(e) de-hybridizing and removing the first fluorescently labeled decoder probe;
(f) hybridizing a second fluorescently labeled decoder probe to said second decoder sequence, wherein the second fluorescently labeled decoder probe that is not hybridized to said second decoder sequence is removed by washing, and wherein the second fluorescently labeled decoder probe is labeled with a fluor that absorbs light at a different wavelength than the fluor of the first fluorescently labeled decoder probe;
(g) detecting a plurality of fluorescent signals indicative of said hybridizing of the second fluorescently labeled decoder probe to said second decoder sequence, and
(h) determining the identity of the padlock probe based upon decoding the plurality of fluorescent signals generated from hybridization of the first fluorescently labeled decoder probe with the first decoder sequence of the padlock probe, and the plurality of fluorescent signals generated from hybridization of the second fluorescently labeled decoder probe to the second decoder sequence of the padlock probe, wherein identification of the padlock probe provides sequence information regarding the at least one target nucleic acid sequence in the plurality of nucleic acid molecules from an individual cell.
2. The method of claim 1 , wherein said amplifying a plurality of nucleic acid molecules in situ within a cell comprises rolling circle amplification.
3. The method of claim 1 , wherein said amplified nucleic acid molecules are immobilized within said cell.
4. The method of claim 1 , wherein said amplified nucleic acid molecules are amplified from cellular RNA.
5. The method of claim 4 , further comprising cDNA synthesis using said cellular RNA.
6. The method of claim 1 , wherein said amplified nucleic acid molecules comprise DNA.
7. The method of claim 1 , wherein said nucleic acid molecules comprise a disease-causing mutation.
8. The method of claim 7 , wherein said disease-causing mutation comprises a base substitution.