Methods for forming adapter ligated nucleic acid molecules
Compositions and methods of use are provided that among other things, allow for efficient adapter ligation to small RNAs. Embodiments of the compositions include partially double stranded polynucleotides for use as 3′ adapters that contain 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 method comprising:
ligating 3′ adaptor molecules to the ends of members of a population of single stranded target polynucleotides, wherein the 3′ adaptor molecules comprise:
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, wherein the random sequences the 3′ adaptor molecules bind to members of the population of single stranded target 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 f removing the single-strand extension by cleavage to form a double-stranded region at t 3′ end of the target polynucleotides;
cleaving the single-strand extensions of the ligated 3′ adaptors;
ligating 5′ adaptors to the 5′ ends of the single stranded target polynucleotides, wherein the 5′ polynucleotide adaptors comprise a top strand and a complementary bottom strand with the bottom strand having a 5′ single-strand extension containing degenerate bases.
2 . The method according to claim 1 , wherein the single stranded target polynucleotides are RNA.
3 . The method according to claim 2 , wherein the polynucleotides are members of a library.
4 . The method according to claim 2 , wherein the polynucleotides are members of a population of RNAs that are variable in size and concentration.
5 . The method according to claim 4 , further comprising, after ligating the 3′ adaptor molecules, cleaving the single-strand extensions of the ligated 3′ adaptors, and ligating 5′ adaptors: reverse transcribing the RNA and forming a cDNA library.
6 . The method according to claim 5 , further comprising: performing the steps of ligating the 3′ adaptor molecules, ligating the 5′adaptors and reverse transcribing the RNA in a one pot workflow.
7 . The method according to claim 5 , wherein a purification step is not performed prior to reverse transcribing the RNA and forming the cDNA library.
8 . The method according to claim 2 , wherein the RNA is sRNA.
9 . The method according to claim 1 , wherein the step of cleaving the single-strand extensions of the ligated 3′ adaptors comprises cleaving a site-specific cleavable sequence or nucleotide in the 3′ adaptors with a nicking restriction endonuclease.
10 . The method according to claim 1 , wherein the step of cleaving the single-strand extensions of the ligated 3′ adaptors comprises cleaving a site-specific cleavable sequence or nucleotide in the 3′ adaptors with a glycosylase/lyase.
11 . The method according to claim 1 , wherein the single stranded target polynucleotides are RNA in a body fluid.
12 . The method according to claim 1 , wherein the single stranded target polynucleotides are RNA in a cell lysate.
13 . The method of claim 1 , wherein cleaving the single-strand extensions of the ligated 3′ adaptors is performed before ligating 5′ adaptors.
14 . The method of claim 1 , wherein cleaving the single-strand extensions of the ligated 3′ adaptors is performed after ligating 5′ adaptors.
15 . The method of claim 1 , wherein the top strand and bottom strand of the 3′ adaptor molecules are formed from two polynucleotide strands.
16 . The method of claim 1 , wherein the top strand and bottom strand of the 3′ adaptor molecules are formed from a single polynucleotide strand.