Multiplex method of preparing a sequencing library

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. One of the forward and reverse tagging primers comprises a purification label at the 5' end, and is provided at a limiting concentration whereby the library constructs comprises an abundance of partial constructs containing only one indexing sequences and only one adapter sequences, and a limited number of full (complete) constructs containing the first and second indexing sequences, the first and second adapter sequences and the purification label. The capture step comprises capturing the full (complete) constructs from the reaction product using the purification label.