Double-stranded splint adaptors with universal long splint strands and methods of use

A library-splint complex (500) comprising:

• a) a single-stranded nucleic acid library molecule (100) comprising from 5′ to 3′:
• (i) a first left universal adaptor sequence (120) having a binding sequence for a first surface primer;
• (ii) a second left universal adaptor sequence (140) having a binding sequence for a first sequencing primer;
• (iii) a sequence of interest (110);
• (iv) a second right universal adaptor sequence (150) having a binding sequence for a second sequencing primer;
• and (v) a first right universal adaptor sequence (130) having a binding sequence for a second surface primer;
• b) a first splint strand (300) comprising from 5′ to 3′:
• a first region (320);
• an internal region (310);
• and a second region (330), wherein the first splint strand comprises at least one abasic site or uracil;
• and
• c) a second splint strand (400) comprising from 3′ to 5′:
• a first sub-region having a universal binding sequence for a third surface primer;
• and a second sub-region having a universal binding sequence for a fourth surface primer;
• wherein the first splint strand (300) is hybridized to portions of the single-stranded nucleic acid library molecule (100) thereby circularizing the single-stranded nucleic acid library molecule to generate a library-splint complex (500), wherein the first region (320) of the first splint strand (300) is hybridized to the binding sequence for the first surface primer, and the third region (330) of the first splint strand (300) is hybridized to the binding sequence for the second surface primer, wherein the second splint strand (400) is hybridized to the internal region (310) of the first splint strand (300), wherein the library-splint complex (500) comprises a first nick between a 5′ end of the single-stranded nucleic acid library molecule and a 3′ end of the second splint strand (400), wherein the library-splint complex (500) comprises a second nick between a 5′ end of the second splint strand (400) and a 3′ end of the single-stranded nucleic acid library molecule.