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. A method of selective extension, comprising:
obtaining a sample comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species;
contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species;
contacting a plurality of oligonucleotide probes with the sample, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a target binding region capable of hybridizing to the plurality of nucleic acid target molecules and the one or more undesirable nucleic acid species, and wherein the blocking oligonucleotide comprises:
(i) a sequence that specifically binds to the at least one of the one or more undesirable nucleic acid species,
(ii) the sequence, or a subsequence, of the target binding region of the oligonucleotide probes, and
(iii) a sequence that does not hybridize to the at least one of the one or more undesirable nucleic acid species; and
extending oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules and the one or more undesirable nucleic acid species to generate a plurality of extension products;
whereby the extension of the at least one of the one or more undesirable nucleic acid species is reduced by the blocking oligonucleotide.
2. The method of claim 1 , wherein the blocking oligonucleotide is contacted with the sample before the plurality of oligonucleotide probes is contacted with the sample.
3. The method of claim 1 , wherein the blocking oligonucleotide is contacted with the sample after the plurality of oligonucleotide probes is contacted with the sample.
4. The method of claim 3 , wherein
obtaining the sample comprises obtaining a second sample comprising the plurality of nucleic acid target molecules and the one or more undesirable nucleic acid species,
contacting the blocking oligonucleotide with the sample comprises contacting the blocking oligonucleotide with the second sample, and/or
contacting the plurality of oligonucleotide probes with the sample comprises contacting the plurality of oligonucleotide probes with the second sample, and
the method comprises pooling the sample and the second sample subsequent to contacting the plurality of oligonucleotide probes with the sample and prior to extending the oligonucleotides that are hybridized to the plurality of nucleic acid target molecules and the one or more undesirable nucleic acid species.
5. The method of claim 1 , wherein the blocking oligonucleotide does not comprise non-natural nucleotides.
6. The method of claim 1 , wherein the blocking oligonucleotide comprises a 3′ non-annealing region configured to not anneal to the one or more undesirable nucleic acid species.
7. The method of claim 1 , 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.
8. The method of claim 1 , wherein the blocking oligonucleotide has a T m of at least 50° C., of at least 60° C., or of at least 70° C.
9. The method of claim 1 , wherein the blocking oligonucleotide is unable to function as a primer for a reverse transcriptase or a polymerase.
10. The method of claim 1 , wherein the extension of the undesirable nucleic acid species is reduced by at least 10%, by at least 25%, by at least 50%, by at least 80%, by at least 90%, by at least 95%, or by at least 99%.
11. The method of claim 1 , wherein the one or more undesirable nucleic acid species amounts to about 50%, to about 60%, to about 70%, or to about 80% of the nucleic acid content of the sample.
12. The method of claim 1 , wherein the undesirable nucleic acid species is selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA, intronic sequence, high abundance sequence, and a combination thereof.
13. The method of claim 1 , wherein each of the plurality of oligonucleotide probes comprises a cellular label sequence, a sample label sequence, a location label sequence, a binding site for a universal primer, or a combination thereof.
14. The method of claim 1 , wherein the plurality of nucleic acid target molecules and/or the undesirable nucleic acid species comprises mRNA molecules and/or DNA molecules.
15. The method of claim 1 , wherein the one or more undesirable nucleic acid species are mRNA molecules and the blocking oligonucleotide specific binds to within 10 nt of the 3′ poly(A) tail of the one or more undesirable nucleic acid species.
16. The method of claim 1 , wherein the target binding region comprises a poly-dT sequence.