Methods for simultaneous amplification of target loci

A method for preparing a DNA fraction from a biological sample of a subject useful for analyzing genetic or epigenetic features associated with cancer, comprising:

• (a) extracting cell-free DNA molecules from the biological sample;
• (b) preparing a DNA fraction by:
• introducing at least 1024 adaptors each containing a universal priming sequence and a different molecular barcode into at least a subset of the extracted cell-free DNA molecules to produce a population of adapted DNA molecules, wherein the at least a subset of the extracted cell-free DNA molecules from the biological sample are tagged with at least 1024 different molecular barcodes, performing universal amplification on at least some of the population of adapted DNA molecules using the universal priming sequence to produce a population of amplified adapted DNA molecules, and selectively enriching for at least 1,000 target loci of interest together in the same reaction volume from at least a subset of the population of amplified adapted DNA molecules or its derivative to produce enriched DNA;
• (c) performing massively parallel sequencing on the enriched DNA or its derivative to obtain sequence reads from at least a portion of the target loci of interest;
• (d) identifying from at least a portion of the sequence reads those derived from a same extracted cell-free DNA molecule in the at least a subset of the extracted cell-free DNA molecules using the tagged molecular barcode and forming grouped sequence reads;
• and
• (e) identifying from at least a plurality of the grouped sequence reads one or more genetic or epigenetic features associated with cancer.