Compositions and methods for preparing nucleic acid libraries
In various aspects, the present disclosure provides methods, compositions, reaction mixtures, kits, and systems for preparing nucleic acid libraries, such as for polynucleotide sequencing. In some embodiments, preparation methods comprise tailing reactions, ligation reactions for attaching an adapter, and an amplification reaction between ligation reactions.
1 . A method for preparing a polynucleotide library, the method comprising, in the following order:
(a) in a first tailing reaction, adding a first tail to each of a plurality of target polynucleotides by template-independent polymerization of a pool of nucleotides consisting of dCTP and dTTP, wherein the dCTP and the dTTP are in a ratio selected from the group consisting of 9:1 and 3:1, such that the first tail has a ratio of dCTP to dTTP selected from the group consisting of 9:1 and 3:1, and the first tail is a single-stranded polynucleotide,
wherein each of the plurality of target polynucleotides comprises a single-stranded polynucleotide,
wherein the first tailing reaction further comprises hybridizing a first adapter to the first tail,
wherein the first adapter comprises a first strand and a second strand forming a double-stranded region and an overhang within the first strand of the first adapter, and
wherein the overhang of the first adapter is hybridized to the first tail;
(b) in a first ligation reaction, ligating one end of the second strand of the first adapter that is on the same side of the overhang in the first adaptor to the end of the first tail;
(c) in a linear amplification reaction, hybridizing a first primer to the second strand of the first adaptor that is ligated to the first tail in (b) and extending the first primer, and repeating the hybridizing and extending for a total of 10 to 15 cycles to linearly amplify each of the plurality of target polynucleotides, thereby producing a plurality of complement target polynucleotides; and
(d) in a second ligation reaction, ligating a strand of a second adapter to the plurality of complement target polynucleotides at the end opposite to the first primer.
2 . The method of claim 1 , wherein (d) comprises (d-1) and (d-2) in the following order,
(d-1) in a second tailing reaction, wherein a second tail is added to each of the plurality of complement target polynucleotides, which are the linear amplification products complementary to the first tail of the plurality of target polynucleotides by template-independent polymerization,
wherein the second tail can have a different sequence from the first tail, depending upon the one or more nucleotides in the nucleotide pool for the second tailing reaction,
wherein the second tail is a single-stranded polynucleotide,
wherein the second tailing reaction further comprises hybridizing the second adapter to the second tail, wherein the second adapter comprises a first strand and a second strand forming a double-stranded region and an overhang within the first strand of the second adapter, wherein the overhang of the second adapter is hybridized to the second tail; and
(d-2) in the second ligation reaction, ligating the second strand of the second adapter to the second tail.
3 . The method of claim 1 , wherein the method further comprises: (i) directly hybridizing the second adapter to each of the plurality of complement target polynucleotides, wherein the second adapter comprising a first strand and a second strand forming a double-stranded region and an overhang within the first strand of the second adapter, wherein the overhang of the second adapter is hybridized to each of the plurality of complement target polynucleotides, and (ii) directly ligating the second strand of the second adapter to the plurality of complement target polynucleotides.
4 . The method of claim 1 , wherein the method further comprises prior to (a) of claim 1 , one or more of: (i) fragmenting polynucleotides to produce the target polynucleotides; (ii) dephosphorylation of one or both ends of the target polynucleotides; and (iii) denaturing double-stranded polynucleotides to single-stranded polynucleotides to produce each of the plurality of target polynucleotides.
5 . The method of claim 4 , further comprising prior to (ii) of claim 4 differentially modifying methylated cytosines or unmethylated cytosines in each of the plurality of target polynucleotides, wherein differentially modifying methylated cytosines or unmethylated cytosines comprises treating each of the plurality of target polynucleotides with bisulfite.
6 . The method of claim 2 , wherein the nucleotide pool for the second tailing reaction consists of one or two types of nucleotides, wherein the one or two types of nucleotides contains or respectively contain DNA bases selected from the group consisting of adenine (A), thymine (T), guanine (G) and cytosine (C).
7 . The method of claim 2 , wherein (i) the first adapter and the second adapter comprise double-stranded regions that are different in polynucleotide sequences, or (ii) the overhang within the first strand of the first adapter or the overhang within the first strand of the second adapter is a 3′-overhang.
8 . The method of claim 7 , wherein the first adapter or the second adapter further comprises a 5′-overhang.
9 . The method of claim 2 , further comprising amplifying the plurality of complement target polynucleotides by extending a second primer hybridized to the second strand of the second adapter that is ligated to the second tail in (d-2) of claim 2 .
10 . The method of claim 9 , further comprising an exponential amplification reaction with a third primer and a fourth primer, wherein (i) the third primer hybridizes to a complement of at least a portion of the first primer, and (ii) the fourth primer hybridizes to a complement of at least a portion of the second primer.
11 . The method of claim 10 , wherein the third primer, the fourth primer, or both the third primer and the fourth primer comprise an index sequence that identifies a sample source of each of the plurality of target polynucleotides.
12 . The method of claim 11 , further comprising:
(e) sequencing the exponential amplification products of the exponential amplification reaction comprising the third primer and the fourth primer; and
(f) grouping sequencing reads according to the index sequence.
13 . The method of claim 12 , wherein sequencing comprises detecting a sequence variant in one or more of the plurality of target polynucleotides or a difference in nucleotide methylation in one or more of the plurality of target polynucleotides, relative to a reference sequence.
14 . The method of claim 2 , wherein the overhang within the first strand of the first adapter consists of nucleotides (G) and (A) in a ratio selected from a group consisting of 9:1 and 3:1.