Methods and reagents for enhanced next generation sequencing library conversion and incorporation of molecular barcodes into targeted and random nucleic acid sequences

A linear double-stranded DNA (dsDNA) Dual Adapter molecule configured to capture or select a specific nucleic acid sequence for targeted sequencing, the Dual Adapter molecule comprising:

• a first homology region (HR1) that comprises a first target homology sequence ranging from a length of 10 nucleotides to 1000 nucleotides that is substantially homologous to a region located 5′ to a predetermined region within a first target DNA, cDNA, and/or RNA molecule, wherein HR1 is comprised of a single stranded 5′ or 3′ protruding end;
• a second homology region (HR2) that comprises a second target homology sequence ranging from a length of 10 nucleotides to 1000 nucleotides that is substantially homologous to a region located 3′ to a predetermined region within the first target DNA, cDNA, and/or RNA molecule, wherein HR2 is comprised of a single stranded 5′ or 3′ protruding end and the first and second homology regions function as a pair (HR1:HR2) and comprise different nucleotide sequences that bind or hybridize to different regions of the same target nucleic acid molecule to form a hybridization complex consisting of the Dual Adapter molecule and target nucleic acid molecule that may be isolated or separated from non-targeted nucleic acids;
• a first unique molecular identifier (UMI1) disposed directly adjacent to HR1 that comprises a random nucleic acid sequence of at least 2 nucleotides;
• a second unique molecular identifier (UMI2) disposed directly adjacent to HR2 that comprises a random nucleic acid sequence of at least 2 nucleotides;
• a first Adapter sequence (AS1) disposed directly adjacent to UMI1;
• a second Adapter sequence (AS2) disposed directly adjacent to UMI2, wherein the first and second Adapter sequences comprise different nucleotide sequences and bind different primers;
• optionally a first tandem restriction or nicking enzyme sequence positioned such that it forms a stem-loop or hairpin structure and can be cleaved or nicked by the corresponding enzyme when a single strand is generated by a DNA polymerase during isothermal amplification or rolling circle amplification and disposed 5′ of, and optionally flanking AS1;
• optionally a second tandem restriction or nicking enzyme sequence positioned such that it forms a stem-loop or hairpin structure and can be cleaved or nicked by the corresponding enzyme when a single strand is generated by a DNA polymerase during isothermal amplification or rolling circle amplification and disposed 3′ of, and optionally flanking AS2;
• optionally at least one or more of the following, (i) a gene encoding a selectable marker, optionally an antibiotic resistance marker, and (ii) an origin of replication.