System and method for detection of HIV tropism variants

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 molecules in an HIV sample population derived from a single patient, wherein each of the cDNA species is generated using a pair of nucleic acid primers that target an HIV V3 region of an HIV clade selected from the group consisting of clade A, clade B, clade C, clade D, and clade G, and wherein the pair of nucleic acid primers are selected from the group consisting of V3-1F (SEQ ID NO:
• 1) and V3-1R (SEQ ID NO:
• 2);
• V3-2F (SEQ ID NO:
• 3) and V3-2R (SEQ ID NO:
• 4);
• V3-1F (SEQ ID NO:
• 1) and V3-2R (SEQ ID NO:
• 4);
• and V3-2F (SEQ ID NO:
• 3) and V3-1R (SEQ ID NO:
• 2);
• (b) amplifying at least one first amplicon from the cDNA species by PCR, wherein each first amplicon comprises a plurality of amplified copies and is amplified with the pair of nucleic acid primers that define a locus of the first amplicon;
• (c) clonally amplifying the amplified copies of the first amplicons by emulsion PCR 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 substrate;
• 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 tropism.