Selective amplification using blocking oligonucleotides
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 amplification, comprising:
providing a sample comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species;
providing a plurality of particles each comprising a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence, a cell label sequence and a binding region, wherein the plurality of oligonucleotide probes comprises at least 100 different molecular label sequences, and wherein oligonucleotide probes associated with the same particle comprise the same cell label sequence and oligonucleotide probes associated with different particles comprise different cell label sequences;
contacting the plurality of oligonucleotide probes with the plurality of nucleic acid target molecules for hybridization;
extending oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules to generate a plurality of extension products;
providing one or more amplification primers;
providing a blocking oligonucleotide that specifically binds to at least one of the one or more undesirable nucleic acid species within 100 nt of the 5′ end of the one or more undesirable nucleic acid species; and
amplifying the plurality of extension products in the presence of the blocking oligonucleotide and one or more amplification primers to generate a plurality of amplicons, wherein the one or more amplification primers add sequencing adaptors to the plurality of extension products,
whereby the amplification of the undesirable nucleic acid species is reduced by the blocking oligonucleotide not being able to function as a primer for a polymerase.
2. The method of claim 1 , wherein the blocking oligonucleotide comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof.
3. The method of claim 1 , comprising providing blocking oligonucleotides that specifically bind to two or more, at least 10, or at least 100 undesirable nucleic acid species in the sample.
4. The method of claim 1 , wherein the blocking oligonucleotide has a T m of at least 60° C., 65° C., or 70° C.
5. The method of claim 1 , wherein the one or more undesirable nucleic acid species amounts to about 50%, 60%, 70%, or 80% of the nucleic acid content of the sample.
6. The method of claim 1 , wherein the undesirable nucleic acid species comprise rRNA, mtRNA, genomic DNA, intronic sequence, high-abundance sequence, or any combination thereof.
7. The method of claim 1 , wherein the blocking oligonucleotide specifically binds to within 50 nt of the 5′ end of the one or more undesirable nucleic acid species.
8. The method of claim 1 , wherein the amplifying comprises PCR amplification of the plurality of extension products.
9. The method of claim 1 , wherein the plurality of oligonucleotide probes comprises at least 1000 or 10000 different molecular label sequences.
10. The method of claim 1 , wherein the plurality of amplicons comprises a cDNA library.
11. The method of claim 1 , wherein the sample comprises a single cell, a plurality of cells, or a tissue sample.
12. The method of claim 1 , further comprising sequencing the plurality of amplicons.
13. The method of claim 1 , wherein the plurality of oligonucleotide probes is immobilized on the particle.
14. The method of claim 1 , wherein the plurality of nucleic acid target molecules comprises mRNA target molecules.
15. The method of claim 1 , wherein the binding region comprises a poly-dT sequence.
16. The method of claim 1 , wherein the plurality of particles comprises streptavidin beads, agarose beads, magnetic beads, protein A-conjugated beads, protein G-conjugated beads, protein A/G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, anti-biotin microbeads, or anti-fluorochrome microbeads.
17. The method of claim 1 , wherein the blocking oligonucleotide specifically binds to within 10 nt of the 5′ end of the one or more undesirable nucleic acid species.
18. A method of selective extension, comprising:
providing a sample comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species;
providing a plurality of particles each comprising a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence, a cell label sequence and a binding region, wherein the plurality of oligonucleotide probes comprises at least 100 different molecular label sequences, and wherein oligonucleotide probes associated with the same particle comprise the same cell label sequence and oligonucleotide probes associated with different particles comprise different cell label sequences;
contacting the plurality of oligonucleotide probes with the plurality of nucleic acid target molecules for hybridization;
providing a blocking oligonucleotide that specifically binds to at least one of the one or more undesirable nucleic acid species within 100 nt of the 5′ end of the one or more undesirable nucleic acid species; and
extending oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules in the presence of the blocking oligonucleotide to generate a plurality of extension products;
whereby the extension of oligonucleotide probes hybridized to the undesirable nucleic acid species is reduced by the blocking oligonucleotide not being able to function as a primer for a polymerase.
19. The method of claim 18 , wherein the blocking oligonucleotide comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof.
20. The method of claim 18 , wherein the plurality of particles comprises streptavidin beads, agarose beads, magnetic beads, protein A-conjugated beads, protein G-conjugated beads, protein A/G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, anti-biotin microbeads, or anti-fluorochrome microbeads.
21. The method of claim 18 , wherein the blocking oligonucleotide specifically binds to within 50 nt of the 5′ end of the one or more undesirable nucleic acid species.
22. The method of claim 18 , wherein the blocking oligonucleotide specifically binds to within 10 nt of the 5′ end of the one or more undesirable nucleic acid species.
23. The method of claim 18 , further comprising sequencing the plurality of extension products, or products thereof, to generate a plurality of sequencing reads.