Efficient methods and compositions for multiplex target amplification PCR

A method of enriching nucleic acid target sequences in a sample, comprising the steps of:

• in a test reaction, hybridizing two or more target-specific primers to nucleic acid target sequences, wherein the target-specific primers comprise a complementary universal auxiliary portion at the 5′-end and a target-specific portion configured to target the nucleic acid target sequences in the sample;
• subjecting the test reaction to a first amplification with universal auxiliary primers under optimal amplification conditions to form an amplified product;
• subjecting a portion of the amplified product to a second amplification using a methylated universal auxiliary primer to form a second amplified product, wherein the methylated universal auxiliary primer comprises a restriction enzyme recognition sequence;
• subjecting the second amplified product to digestion with a methylation-dependent endonuclease restriction enzyme to form a digestion product comprising amplicons comprising sticky ends on each end of the strands;
• performing size selection purification on the digestion product to remove digested primer-dimers and unused primers to form digested amplicons comprising dsDNA;
• ligating universal adapters to dsDNA from the digested amplicons to form a ligated product, wherein the ligating universal adaptors comprise complementary sticky ends and a universal sequence portion;
• and
• subjecting the ligated product to a third amplification using barcoded universal primers complementary to sequences on the ligating universal adapters to form a final amplification product comprising final amplicons.