Methods, compositions, and kits for inhibiting formation of adapter dimers

A pair of oligonucleotides for reducing adapter-dimers in a sequencing library, 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;
• 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.