Quantitative nuclease protection assay (qnpa) and sequencing (qnps) improvements

A method of detecting at least one target nucleic acid molecule in a sample, comprising:

• contacting the sample with at least one nuclease protection probe comprising a flanking sequence (NPPF) under conditions sufficient for the NPPF to specifically bind to the target nucleic acid molecule,
• wherein the NPPF comprises:
• a 5′-end and a 3′-end,
• a sequence complementary to a region of the target nucleic acid molecule, permitting specific binding between the NPPF and the target nucleic acid molecule,
• a flanking sequence located 5′, 3′, or both, to the sequence complementary to the target nucleic acid molecule, wherein the flanking sequence comprises at least 12 contiguous nucleotides not found in a nucleic acid molecule present in the sample providing a universal amplification sequence, and wherein the flanking sequence is complementary to at least a portion of an amplification primer;
• contacting the sample with a nucleic acid molecule comprising a sequence complementary to the flanking sequence (CFS) under conditions sufficient for the flanking sequence to specifically bind to the CFS;
• contacting the sample with a nuclease specific for single-stranded nucleic acid molecules under conditions sufficient to remove unbound nucleic acid molecules, thereby generating a digested sample comprising NPPFs hybridized to the target nucleic acid molecule and to the CFS(s);
• amplifying NPPFs in the digested sample with amplification primer, thereby generating NPPF amplicons;
• and
• detecting the NPPF amplicons, thereby detecting the at least one target nucleic acid molecule in the sample.