Patent Document ID: 9951380
Application ID: 14795774
Patent Flag: 1

Claim One:
1. A method of generating an amplicon library from genomic DNA comprising: a) contacting a portion of a nucleic acid sample with a plurality of different target-specific primers, and a polymerase under amplification conditions and producing a plurality of different target-specific amplicons by extending one or more of the plurality of target-specific primers; b) digesting at least one of the plurality of different target-specific primers with a cleaving reagent; and c) ligating an adapter to the end of the at least one different target-specific amplicon and producing at least one adapter-ligated target-specific amplicon; wherein each of the plurality of target specific primers have the following criteria: (1) includes two or more modified cleavable nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence; (2) length is about 15 to about 40 bases in length; (3) T m is from about 60° C. to about 70° C.; (4) has low cross-reactivity with non-target sequences present in the sample; (5) at least the first four nucleotides (going from 3′ to 5′ direction) are non-complementary to any sequence within any other primer present in the reaction; and (6) are non-complementary to any consecutive stretch of at least 5 nucleotides within any other produced amplified target sequence; and wherein none of the adapters in a ligation mixture, prior to ligating, can hybridize under high stringency, to any one of the target specific amplicon sequences in the reaction; and wherein the method is optionally carried out in a single reaction, addition only process.