Polynucleotide adapter design for reduced bias
Compositions are provided for 3′ adapters and methods of use are provided that include methods requiring a plurality of ligation steps involving a single-stranded target polynucleotide and 3′ and 5′ adapters. Embodiments of the 3′ adapters comprise a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3′ adapters, the single-stranded region is released by cleaving the cleavable linker.
1. A composition comprising: a partially double-stranded polynucleotide molecule having a top strand and a bottom strand, wherein:
(a) the top strand comprises a nucleic acid sequence that is complementary to a portion of the nucleic acid sequence of the bottom strand, such that the top strand and bottom strand form a double-stranded region by complementary base-pairing; and
(b) the bottom strand comprises: (i) a non-complementary 3′ single-stranded extension, (ii) a sequence of at least 4 degenerate nucleotides, wherein the at least 4 degenerate nucleotide sequence is a random sequence that differs for each polynucleotide in a population of polynucleotides; and (iii) a site-specific cleavable sequence or nucleotide at or near the junction between the double-stranded region and the single-strand extension, suitable for removing the single-strand extension by cleavage;
wherein the partially double-stranded polynucleotide molecule is suitable for use as a 3′ polynucleotide adapter.
2. The composition according to claim 1 , wherein the bottom strand further comprises a blocking moiety at the 3′ terminus.
3. The composition according to claim 1 , wherein the top strand has a phosphorylated or pre-adenylated 5′ terminus.
4. The composition according to claim 1 , wherein the partially double-stranded polynucleotide is formed from a polynucleotide that is single-stranded.
5. The composition according to claim 1 , wherein the partially double-stranded polynucleotide is formed from two polynucleotide strands.
6. The polynucleotide molecule of claim 1 , wherein the polynucleotide molecule is a DNA.
7. The polynucleotide molecule of claim 1 , wherein the polynucleotide molecule is RNA.
8. The polynucleotide molecule of claim 1 , wherein the 3′ single-stranded extension has a length in the range of 4-12 nucleotides.
9. The polynucleotide molecule of claim 1 , wherein the site-specific cleavable sequence or nucleotide is a deoxyuridine.
10. The polynucleotide molecule of claim 1 , wherein the site-specific cleavable sequence or nucleotide is a restriction endonuclease cleavage site.
11. The polynucleotide molecule of claim 2 , wherein the blocking nucleotide prevents ligation.
12. The polynucleotide according to claim 2 , wherein the blocking moiety comprises a modification selected from the group consisting of a 3′ inverted dT, a 3′ C3 spacer, a 3′ amino dN, a 3′ phosphorylated dN, and a dideoxynucleotide.
13. The polynucleotide molecule of claim 1 , wherein the site-specific cleavable sequence or nucleotide is positioned at the junction of the single-stranded extension and the double-stranded region.
14. The polynucleotide molecule of claim 1 , wherein the site-specific cleavable sequence or nucleotide is positioned within the double-stranded region on the bottom strand within 8 nucleotides of the junction of the single-stranded extension and the double-stranded region.
15. The polynucleotide according to claim 1 , wherein there is more than one cleavable nucleotide or sequence in the polynucleotide molecule, wherein each of the cleavable nucleotides or sequences is positioned in the double-stranded region on the bottom strand within 8 nucleotides of the junction of the single-stranded extension and the double-stranded region.
16. A kit comprising:
(a) a partially double-stranded polynucleotide molecule according to claim 1 ; and
(b) a second polynucleotide molecule comprising a top strand and a bottom strand, wherein the top strand comprises or consists of a nucleic acid sequence that is complementary to a portion of the nucleic acid sequence of the bottom strand, such that the top strand and bottom strand form a double-stranded region by complementary base-pairing, and wherein the bottom strand comprises a 5′ single-stranded extension with a sequence containing at least 4 degenerate nucleotides, wherein the sequence containing the at least 4 degenerate nucleotide sequence is a random sequence that differs for each polynucleotide in a population of polynucleotides.
17. The kit according to claim 16 , further comprising one or more enzymes selected from the group consisting of a ligase, a nicking endonuclease, a glycosylase, a deadenylase, and an exonuclease.
18. A kit comprising:
(a) a polynucleotide molecule according to claim 1 , for use as a 3′ adapter; and
(b) one or more enzymes selected from the group consisting of a ligase, a nicking endonuclease, a glycosylase, a deadenylase, and an exonuclease.
19. The kit according to claim 18 , further comprising a second polynucleotide molecule for use as a 5′ adapter, comprising: a top strand and a bottom strand, wherein (i) the top strand comprises a nucleic acid sequence that is complementary to a portion of the nucleic acid sequence of the bottom strand, (ii) the bottom strand comprises a 5′ single-stranded extension a sequence of at least 4 degenerate nucleotides and optionally a blocking moiety at the 5′ terminus, wherein the at least 4 degenerate nucleotide sequence is a random sequence that differs for each polynucleotide in a population of polynucleotides; (iii) a site-specific cleavable sequence or nucleotide at or near the junction between the double-stranded region and the single-strand extension, suitable for removing the single-strand extension by cleavage, and optionally; (iv) the first polynucleotide molecule is DNA for ligating to a 5′ end of a target polynucleotide and the second polynucleotide molecule is an RNA for ligating to the 5′ end of the target polynucleotide molecule.
20. A method ligating a 3′ polynucleotide adapter to a population of target polynucleotides, comprising:
(a) using the polynucleotide molecule of claim 1 as a 3′ adapter;
(b) combining the 3′ adapter with a population of target polynucleotides to produce a reaction mix;
(c) incubating the reaction mix to ligate the 3′ adapter to the 3′ of the target polynucleotides; and
(d) cleaving the 3′ adapter at the site-specific cleavable sequence or nucleotide after step (c) so as to remove the degenerate sequence.
21. The method according to claim 20 , wherein the 3′ adapter is DNA and the target polynucleotide is RNA.
22. The method of claim 21 , further comprising:
(e) adding a 5′ adapter molecule having a 5′ single-strand extension comprising degenerate nucleotides to the product of step (c) to produce a second reaction mix; and
(f) incubating the second reaction mix to ligate the 5′ polynucleotide adapter to the RNA molecules.
23. The method according to claim 22 , wherein the 5′ adapter is RNA.
24. The method according to claim 22 , wherein steps (a)(f) are performed in a single reaction vessel.
25. The method according to claim 22 , wherein no intermediate purification or separation steps are performed between steps (a)-(f).
26. The method according to claim 20 , further comprising incubating the product of step (f) with a reverse transcriptase, to copy the ligated RNA into cDNA.
27. The method according to claim 26 , wherein cDNA synthesis is primed using the bottom strand of the polynucleotide molecule, after the 3′ single-stranded extension has been cleaved.
28. The method according to claim 20 , wherein adapter ligation yield and bias does not vary significantly for other populations of RNA.
29. The method according to claim 20 , wherein the RNA molecules are variable in size and concentration.
30. A partially double-stranded polynucleotide molecule comprising a double-stranded region having a first nucleic acid strand and a second complementary nucleic acid strand, wherein:
(i) the first and second strands are a portion of one or comprise 2 polynucleotide molecules;
(ii) the first nucleic acid strand optionally comprises one or more of a phosphorylated or pre-adenylated at the 5′ terminus and a blocking moiety at the 3′ terminus;
(iii) the second complementary strand having a nucleic acid sequence that extends 3′ from the double-stranded region to form a single-stranded extension containing at least 4 degenerate nucleotides in a sequence that wherein the sequence differs for each polynucleotide in a population of polynucleotides; and
(iv) a site-specific cleavable sequence or nucleotide at or near the junction between the double-stranded region and the single-strand extension, suitable for removing the single-strand extension by cleavage.