PCR-free library preparation using double-stranded splint adaptors and methods of use

A method for forming a plurality of library-splint complexes (500) comprising:

• a) providing a plurality of double-stranded splint adaptors (200), wherein individual double-stranded splint adaptors (200) in the plurality comprise a first splint strand (300) hybridized to all or a portion of a second splint strand (400),
• wherein the double-stranded splint adaptor includes a double-stranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region (320), an internal region (310) comprising a universal adaptor sequence for a surface capture primer binding site, and a second region (330), and wherein:
• ii) the internal region of the first splint strand (310) is hybridized to all or a portion of the second splint strand (400), and
• (ii) the internal region (310) of the first splint strand (300) comprises at least three sub-regions comprising sub-region (311), sub-region (312) and sub-region (313),
• wherein the first splint strand, or both splint strands comprise sample index sequence comprising a 3-mer random sequence (NNN), and
• wherein sub-region (311), subregion (312) or subregion (313) comprise the sample index sequence of the first splint strand, and the sample index sequence of the first splint strand comprises an 18-carbon spacer and/or an 18-carbon spacer and at least one deoxyinosine;
• and
• b) hybridizing the plurality of double-stranded splint adaptors with a plurality of single-stranded nucleic acid library molecules (100), wherein individual single-stranded nucleic acid library molecules comprise a sequence of interest (110) flanked on a first side by a universal adaptor sequence for a forward sequencing primer binding site (120) and flanked on a second side by a universal adaptor sequence for a reverse sequencing primer binding site (130), wherein the first region of the first splint strand (320) hybridizes to the universal adaptor sequence for the forward sequencing primer binding site (120) and the second region of the first splint strand (330) hybridizes to the universal adaptor sequence for the reverse sequencing primer binding site (130), wherein the first splint strand (300) comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NOS:
• 7-10 and 12-14,
• thereby circularizing the plurality of single-stranded nucleic acid library molecules to form a plurality of library-splint complexes (500), individual library-splint complexes having two nicks.