Methods for simultaneous amplification of target loci

A method for preparing a fraction of DNA useful for analyzing cancer-associated loci from an individual, comprising:

• (a) extracting DNA from a biological sample from the individual to obtain a sample of cell-free DNA (cfDNA);
• (b) producing a fraction of the DNA extracted in (a) by amplifying at least 10 target loci relating to cancer-associated mutations to obtain amplification products that comprise the at least 10 target loci, wherein the amplifying comprises:
• (1) attaching adaptors to the cfDNA to produce adaptor-attached cfDNA, wherein the adaptors comprise universal priming sequences,
• (2) a first PCR amplifying the at least 10 target loci comprising contacting the adaptor-attached cfDNA with a first universal primer and with at least 10 non-identical target specific primers in a single reaction volume, and
• (3) a second, nested PCR amplifying the at least 10 target loci amplified in (2) comprising contacting the reaction products from (2) with a second universal primer and with at least 10 non-identical inner target specific primers in a single reaction volume, wherein the at least 10 target loci are single nucleotide variant (SNV) loci;
• and
• (c) analyzing the at least 10 target loci in the fraction of DNA produced in (b).