Abstract: Oligonucleotide pairs for reducing adapter-dimers in a sequencing library according to aspects of the present disclosure include: a first and a second blocker oligonucleotide, each comprising: i) a blocker oligonucleotide 5? portion at least 8 nucleotides in length and ii) a 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; wherein the first blocker oligonucleotide 3? portion and the second blocker oligonucleotide 3? portion are equal in length and complementary to each other; and wherein the first blocker oligonucleotide 5? portion and the second blocker oligonucleotide 5? portion are both complementary to the 5? end portion of the 3? adapters.

Abstract: 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.

Abstract: 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 the 5? terminus of a first unligated 3? adapter and a second blocker oligonucleotide 3? terminus adjacent to an adenylated nucleotide at the 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.