Abstract: A method for obtaining a normalized quantity of DNA amplicons includes adding to a volume of a sample comprising a target DNA segment, a first concentration of a forward primer. The method includes adding, to the volume of the sample, a second concentration of a second primer, wherein the second primer comprises an unlabeled reverse primer. The method includes adding a third concentration of the third primer, wherein the third primer is a ligand modified reverse primer complementary to a second end of the target DNA segment and comprises a normalizing ligand. The method further includes performing a polymerase chain reaction (PCR) on the volume of the sample to obtain a normalized concentration of ligand-tagged amplicons, wherein the PCR comprises one or more melt cycles, annealing cycles, and extension cycles.

Abstract: A method of preparing a library of library constructs by multiplex amplification for use in targeted next generation sequencing is described. The method comprises the steps of: (a) providing a reaction vessel comprising (i) a plurality of different target sequences, (ii) a plurality of target capture primer pairs, and (iii) one or more tagging primer pairs, (b) performing sequential rounds of amplification at sequential annealing temperatures configured to amplify the target sequences, generate target sequences comprising first or second read sequences, and provide a reaction product comprising library constructs in a sequential manner; and (c) capture of the library of constructs from the reaction product.