Analysis of heterogeneous viral populations by direct DNA sequencing

The ability of direct PCR sequencing to detect and quantify sequence polymorphisms was investigated using samples containing mixed populations of HIV-1. A part of the genome encoding the polymorphic variable region 3 of the envelope was directly sequenced to yield a consensus sequence of the virus population. The results were compared with sequences obtained by analysis of multiple clones derived from the same clinical samples. The results of five patients suggested that the direct-sequencing method can be used as a rapid tool to analyze and quantify heterogeneous viral populations. Reconstitution experiments using cloned material demonstrated that it was possible to detect and quantify minor sequence variants present in as little as 10% of the total virus population. The use of the method for molecular diagnosis is discussed.