Methods and compositions for mitigating index hopping in dna sequencing

A method of indexing at least one nucleic acid sample, comprising the steps of:

• for each sample, hybridizing a plurality of indexed target-specific primers with the sample in the presence of indexed universal primers to form a plurality of test reactions in a single reaction container, wherein at least one target-specific primer is configured to bind to at least one target nucleic acid sequence and wherein nucleic acid targets are first labeled with indexed target-specific primers in primer extensions of amplification;
• simultaneously amplifying the index-labeled products using indexed universal primers, wherein each test reaction is subject to amplification conditions configured to generate amplicons containing four combinatorial indexes, wherein one index (A) is located at the 5′ end of amplicons at Watson (forward) strand, one index (B) is located at the 5′ end of amplicons at Crick (reverse) strand, one index (X) is located adjacent to target-specific primers at the Watson (forward) strand, and one index (Y) is located adjacent to target-specific primers at the Crick (reverse) strand;
• and
• subjecting at least a portion of the pooled amplicons to next-generation sequencing.