Nucleic acid detection and quantification

A method comprising

• a) ligating adaptors to DNA fragments to produce a population of library adaptor ligation products
• b) combining the population of library adaptor ligation products with
• i.) an intercalating dye recognizing double-stranded DNA molecules with higher affinity than single-stranded DNA molecules and said intercalating dye has increasing fluorescence upon intercalation with double-stranded nucleic acid molecules, and
• ii.) at least one fluorophore-labeled oligonucleotide-probe being sequence specific for one or more target nucleic acid molecules, and said at least one fluorophore-labeled oligonucleotide-probe undergoes a detectable change in fluorescence upon amplification of the one or more target nucleic acid molecules, in a quantitative real time polymerase chain reaction;
• c) incubating the mixture of step (b) with a DNA polymerase under conditions sufficient to amplify one or more of the population of library adaptor ligation products, wherein amplification results in a sequencing library comprising one or more amplified library adaptor ligation products;
• d) detecting the presence or absence or quantifying the amount of the amplified one or more sequencing library adaptor ligation products by measuring fluorescence of said intercalating dye and of said fluorophore;
• and
• e) determining, based on the fluorescence of intercalating dye and fluorophore detected in step d), the percentage of effective sequencing library in the total population of library adaptor ligation products and the efficiency of sequencing library construction or determining the percentage of effective sequencing library in the total population of library adaptor ligation products and simultaneously determining the quantity and quality of the sequencing library.