Patent ID: 8501409

Claim:
A method for detecting low frequency occurrence of one or more HIV sequence variants associated with drug resistance comprising the steps of: (a) generating a plurality of cDNA species from a plurality of RNA molecule in an HIV sample; (b) amplifying a plurality of first amplicons from the cDNA species, wherein each first amplicon comprises a plurality of amplified copies and is amplified with a pair of nucleic acid primers specific to Clade B that define a locus of the first amplicon population wherein the pair of nucleic acid primers are selected from the group consisting of B-ACF-1 (SeqID No: 39) and B-AR (SeqID No: 36); B-ACF-2 (SeqID No: 40) and B-AR (SeqID No: 36); B-BF (SeqID No: 35) and B-BR (SeqID No: 38); B-ACF-1 (SeqID No: 39) and B-CR (SeqID No: 37); B-ACF-2 (SeqID No: 40) and B-CR (SeqID No: 37); B-1F (SeqID No: 42) and B-1R-1 (SeqID No: 44); B-1F (SeqID No: 42) and B-1R-2 (SeqID No: 45); B-2F (SeqID No: 43) and B-2R (SeqID No: 49); B-3F (SeqID No: 50) and B-3R (SeqID No: 51); B-4F (SeqID No: 41) and B-4R-1 (SeqID No: 46); B-4F (SeqID No: 41) and B-4R-2 (SeqID No: 47); B-5F (SeqID No: 48) and B-5R-1 (SeqID No: 52); and B-5F (SeqID No: 48) and B-5R-2 (SeqID No: 53); (c) clonally amplifying the amplified copies of the first amplicons to produce a plurality of second amplicons wherein a plurality of the second amplicons comprise an immobilized population of substantially identical copies from one of the amplified copies of first amplicons; (d) determining a nucleic acid sequence composition of the substantially identical copies from at least 100 of the immobilized populations in parallel on a single instrument; and (e) detecting one or more sequence variants that occur at a frequency of 5% or less in the nucleic acid sequence composition of the at least 100 immobilized populations; and (f) correlating the detected sequence variants with variation associated with HIV drug resistance.