Methods, compositions, and kits for inhibiting formation of adapter dimers
Methods of reducing adapter-dimers in a sequencing library are provided according to aspects of the present disclosure, including: hybridizing excess 3′ adapters to blocker oligonucleotides forming a hybridized complex, such that the excess 3′ adapters are unavailable to form adapter-dimers, the hybridized complex including: a first blocker oligonucleotide 3′ terminus adjacent to an adenylated nucleotide at 5′ terminus of a first unligated 3′ adapter and a second blocker oligonucleotide 3′ terminus adjacent to an adenylated nucleotide at 5′ terminus of a second unligated 3′ adapter, wherein the first blocker oligonucleotide has a 5′ portion complementary to and hybridized to the first unligated 3′ adapter, the second blocker oligonucleotide has a 5′ portion complementary to and hybridized to the second unligated 3′ adapter, and the first blocker oligonucleotide has a 3′ portion complementary to, and hybridized to, a 3′ portion of the second blocker oligonucleotide.
1 . A method of reducing adapter-dimers in a sequencing library, comprising:
combining sample nucleic acids, each having a 5′ end and a 3′ end, with 3′ adapters each having a 5′ end portion at least 8 nucleotides in length and a 5′ terminus which is an adenylated nucleotide, and a ligase, in a mixture under ligation reaction conditions, producing 1) first ligation products each comprising 3′ end of a sample nucleic acid ligated to the 5′ terminus of a 3′ adapter, and 2) excess unligated, 3′ adapters in the mixture;
adding blocker oligonucleotides to the mixture, the blocker oligonucleotides comprising:
a) a first blocker oligonucleotide comprising:
i) a first blocker oligonucleotide 5′ portion at least 8 nucleotides in length and ii) a first blocker oligonucleotide 3′ portion at least 1 to 20 nucleotides in length and comprising a first oligonucleotide 3′ terminus, wherein at least 1 of the 1 to 20 nucleotides is a non-naturally occurring nucleotide analog; and
b) a second blocker oligonucleotide comprising:
iii) a second oligonucleotide 5′ portion at least 8 nucleotides in length and iv) a second blocker oligonucleotide 3′ portion at least 1 to 20 nucleotides in length and comprising a second blocker oligonucleotide 3′ terminus, wherein at least 1 of the 1 to 20 nucleotides is a non-naturally occurring nucleotide analog;
wherein the first blocker oligonucleotide 3′ portion and the second blocker oligonucleotide 3′ portion are equal in length and complementary to each other;
wherein the first blocker oligonucleotide 5′ portion and the second blocker oligonucleotide 5′ portion are both complementary to 5′ end portion of 3′ adapters;
incubating the mixture under hybridization conditions, thereby hybridizing the first blocker oligonucleotide 3′ portion with the second blocker oligonucleotide 3′ portion, hybridizing the first blocker oligonucleotide 5′ portion with the excess unligated 3′ adapters, and thereby hybridizing the second blocker oligonucleotide 5′ portion with the excess unligated 3′ adapters, producing a hybridized complex wherein the first blocker oligonucleotide 3′ terminus is adjacent to an adenylated 5′ end nucleotide of a first unligated 3′ adapter and the second blocker oligonucleotide 3′ terminus is adjacent to an adenylated 5′ end nucleotide of a second unligated 3′ adapter; thereby reducing excess unligated 3′ adapters in the mixture, producing a purified mixture.
2 . The method of claim 1 , wherein the first blocker oligonucleotide 3′ terminus and the second blocker oligonucleotide 3′ terminus are both capable of being ligated to the adenylated nucleotide at the 5′ terminus of the excess unligated 3′ adapters; and further comprising ligating the first blocker oligonucleotide 3′ terminus of the hybridized complex to the adjacent adenylated nucleotide at the 5′ terminus of the first unligated 3′ adapter and ligating the second blocker oligonucleotide 3′ terminus of the hybridized complex to the adjacent adenylated nucleotide at the 5′ terminus of the second unligated 3′ adapter, producing ligated complex, thereby reducing excess unligated 3′ adapters in the mixture, producing a purified mixture.
3 . The method of claim 1 , wherein 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the at least 1 to 20 nucleotides of the first blocker oligonucleotide 3′ portion are non-naturally occurring nucleotide analogs and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the at least 1 to 20 nucleotides of the second blocker oligonucleotide 3′ portion are non-naturally occurring nucleotide analogs.
4 . The method of claim 1 , wherein when the first blocker oligonucleotide 3′ portion and the second blocker oligonucleotide 3′ portion are each 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, the first blocker oligonucleotide 3′ portion and the second blocker oligonucleotide 3′ portion each comprise a palindromic nucleotide sequence, wherein the palindromic nucleotide sequence of the first blocker oligonucleotide 3′ portion is identical to the palindromic nucleotide sequence of the second blocker oligonucleotide 3′ portion.
5 . The method of claim 1 , further comprising: adding 5′ adapter to the mixture, the 5′ adapter having a 3′ terminus, wherein the first ligation products each comprise 3′ end of a sample nucleic acid ligated to the 5′ terminus of a 3′ adapter, and 5′ end of a sample nucleic acid ligated to the 3′ terminus of a 5′ adapter, thereby producing sequencing library molecules, the sequencing library molecules comprising a sample nucleic acid insert disposed between, and
bonded to, a 5′ adapter and a 3′ adapter.
6 . The method of claim 1 , further comprising: adding 5′ adapter to the purified mixture; and incubating the purified mixture under ligation reaction conditions, thereby 1) ligating the 5′ adapter to the first ligation product, producing sequencing library molecules, the sequencing library molecules comprising a sample nucleic acid insert disposed between, and bonded to, a 5′ adapter and a 3′ adapter.
7 . The method of claim 6 , wherein excess unligated 3′ adapters are reduced but not eliminated such that the ligating includes ligating 5′ adapter to at least some of the excess unligated 3′ adapter, producing adapter-dimers comprising a 5′ adapter bonded to a 3′ adapter at a 5′ adapter-3′ adapter interface; and further comprising inhibiting amplification of the adapter-dimers in an amplification reaction such that the sequencing library molecules are selectively enriched by the amplification reaction compared to the adapter-dimers.
8 . The method of claim 7 , wherein inhibiting amplification of the adapter-dimers comprises removing at least some of the adapter-dimers prior to amplification.
9 . The method of claim 7 , wherein inhibiting amplification of the adapter-dimers comprises one or more of: specific enzymatic cleavage of the adapter-dimers; CRISPR/Cas9-based cleavage of the adapter-dimers; gel purification; SPRI-bead based size selection; and adding an adapter-dimer blocker oligonucleotide to the mixture, the adapter-dimer blocker oligonucleotide effective to inhibit amplification of the adapter-dimers.
10 . The method of claim 9 , wherein the adapter-dimer blocker oligonucleotide is complementary to one or more adjacent portions of the adapter-dimers, the one or more adjacent portions comprising at least one nucleotide of 5′ adapter at 5′ adapter-3′ adapter interface, and an adjacent portion of 3′ adapter comprising an amplification primer binding site.
11 . The method of claim 1 , wherein the first blocker oligonucleotide and the second blocker oligonucleotide have the same nucleotide sequence.
12 . The method of claim 1 , wherein the sample nucleic acids are, or include, RNA, and
further comprising reverse transcribing the RNA, producing reverse transcribed sequencing library molecules.
13 . The method of claim 1 , wherein the sample nucleic acids are, or include, DNA.
14 . The method of claim 12 , further comprising amplifying the sequencing library molecules.