SELECTIVE EXTENSION IN SINGLE CELL WHOLE TRANSCRIPTOME ANALYSIS
Disclosed herein include methods and compositions for selectively amplifying and/or extending nucleic acid target molecules in a sample. The methods and compositions can, for example, reduce the amplification and/or extension of undesirable nucleic acid species in the sample, and/or allow selective removal of undesirable nucleic acid species in the sample.
1 - 16 . (canceled)
17 . A kit for selective amplification of nucleic acid molecules in a sample, comprising:
a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a target binding region comprising a poly-dT sequence; and
a plurality of blocking oligonucleotides that specifically binds to a plurality of undesirable mRNA species in the sample, wherein the plurality of blocking oligonucleotides binds to the non-poly(A) region of the plurality of undesirable mRNA species, and wherein each blocking oligonucleotide probe is unable to function as a primer for a reverse transcriptase or a polymerase.
18 . The kit of claim 17 , wherein at least one of the plurality of oligonucleotide probes comprises one of the plurality of blocking oligonucleotides.
19 . The kit of claim 17 , wherein one of the plurality of blocking oligonucleotides comprises (i) a sequence that specifically binds to at least one of the plurality of undesirable nucleic acid species, and (ii) a poly-dT sequence, and/or (iii) a sequence that does not hybridize to the at least one of the plurality of undesirable nucleic acid species.
20 . The kit of claim 17 , wherein the blocking oligonucleotide comprises a 3′ non-annealing region configured to not anneal to the one or more undesirable nucleic acid species.
21 . The kit of claim 20 , wherein the non-complementarity between the 3′ non-annealing region and the region of the undesirable nucleic acid species 5′ adjacent to the sequence specifically bound by the blocking oligonucleotide is at least 50%, is at least 60%, is at least 70%, is at least 80%, is at least 90%, is at least 95%, or is about 100%.
22 . The kit of claim 17 , wherein the blocking oligonucleotide is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, or a PNA/DNA chimera.
23 . The kit of claim 17 , wherein the plurality of oligonucleotide probes is immobilized on a particle.
24 . The kit of claim 17 , wherein each blocking oligonucleotide probe has a T m of at least 60° C.
25 . The kit of claim 17 , wherein the blocking oligonucleotide does not comprise non-natural nucleotides.
26 . The kit of claim 19 , wherein the poly-dT sequence of the oligonucleotide probe is longer than the poly-dT sequence of the blocking oligonucleotide.
27 . The kit of claim 19 , wherein the poly-dT sequence of the oligonucleotide probe and the poly-dT sequence of the blocking oligonucleotide have an identical length.
28 . The kit of claim 17 , wherein none of the plurality of oligonucleotide probes comprises the blocking oligonucleotide.
29 . The kit of claim 20 , wherein the 3′ non-annealing region is 1 nt to 100 nt long, is 1 nt to 50 nt long, is 1 nt to 21 nt long, is 1 nt to 10 nt long, or is about 5 nt long.
30 . The kit of claim 17 , wherein the plurality of blocking oligonucleotides specifically binds to two or more undesirable nucleic acid species.
31 . The kit of claim 17 , wherein the blocking oligonucleotide is 8 nt to 100 nt long.
32 . The kit of claim 17 , wherein the plurality of undesirable mRNA species comprises ribosome mRNA, mitochondrial mRNA, or a combination thereof.
33 . The kit of claim 17 , wherein the blocking oligonucleotides specifically bind to within 100 nt of the 3′ end of the undesirable nucleic acid species.
34 . The kit of claim 17 , wherein the plurality of oligonucleotide probes are immobilized on a substrate, and wherein the substrate is a particle.
35 . The kit of claim 17 , further comprising an enzyme selected from the group consisting of a reverse transcriptase, a polymerase, a ligase, a nuclease, and a combination thereof.
36 . The kit of claim 17 , wherein the plurality of blocking oligonucleotides specifically binds to at least 50 undesirable nucleic acid species.