Methods of library construction for polynucleotide sequencing
Disclosed herein are methods and compositions for the detection of small RNAs in a sample. The methods and compositions disclosed herein may be used for preparing sequencing libraries of the small RNAs, fragments of RNAs and DNAs.
1. An adapter in the form of a single-stranded nucleic acid comprising:
a) a single-stranded 5′-proximal segment and a single-stranded 3′-proximal segment, wherein the single-stranded 5′-proximal segment and the single-stranded 3′ proximal segment comprise a sequencing adapter, a detection sequence, or a combination thereof, wherein the adaptor comprises at least one DNA residue and at least one RNA residue;
b) 5′-end and 3′-end groups that allow first and second consecutive ligation reactions either directly or after conversion of one or both 5′-end and 3′-end groups to ligatable 5′-end and ligatable 3′-end group(s); and
c) a template-deficient segment linking the single-stranded 5′ proximal segment and the single-stranded 3′ proximal segment that restricts primer extension by a polymerase over the template-deficient segment, wherein the template-deficient segment is a non-nucleotide segment.
2. The adapter of claim 1 , wherein the adapter comprises a modified nucleic acid residue.
3. The adapter of claim 1 , wherein the adapter comprises a sequence selected from: a sequencing adapter, a primer binding site, a detection sequence, a probe hybridization sequence, a capture oligonucleotide binding site, a polymerase binding site, an endonuclease restriction site, a sequencing bar-code, an indexing sequence, a Zip-code, one or more random nucleotides, a unique molecular identifier (UMI), and a sequencing flow-cell binding site, and combinations thereof.
4. The adapter of claim 1 , wherein the 5′-end or 3′-end of the adapter contains a reversible blocking group preventing circularization or concatamerization of the adapter while allowing its ligation with a sample polynucleotide, wherein the reversible blocking group requires chemical, photochemical or enzymatic conversion to active end group prior to the circularization of the adapter-polynucleotide ligation product.
5. The adapter of claim 4 , wherein the reversible blocking group is a 3′-end-blocking group selected from: 3′-p, 2′,3′-cyclic phosphate (2′,3′>p), 3′-O-(α-methoxyethyl)ether, and 3′-O-isovaleryl ester.
6. A kit for preparing an RNA sequencing library comprising the adapter according to claim 1 .
7. A plurality of the single stranded nucleic acid of claim 1 .
8. The adapter of claim 1 , wherein the non-nucleotide residue comprises a non-nucleotide residue selected from: an abasic site, a disulfide (S—S) internucleotide bond, and combinations thereof.
9. The adapter of claim 1 , wherein the single-stranded 5′-proximal segment comprises at least one RNA residue and the single-stranded 3′-proximal segment comprises at least one DNA residue.
10. The adapter of claim 1 , wherein the single-stranded 3′-proximal segment comprises at least one RNA residue and the single-stranded 5′-proximal segment comprises at least one DNA residue.