Relative quantitation of virus population size in mixed genotype infections using sequencing chromatograms

In order to quantitatively distinguish between highly similar RNA sequences, specific primers or probes must be designed. Unfortunately, consistent and reliable results are not always obtained with conventional techniques. This study uses reverse transcription-PCR coupled with direct terminator sequencing to economically and efficiently distinguish between sequence types in pooled samples while providing accurate relative quantification. As an example, the method is applied to measure template concentration of two Barley yellow dwarf virus (BYDV; family Luteoviridae) species in doubly infected wheat plants. A PERL script (polySNP) was developed that uses PHRED to automatically extract relative peak areas and heights from sequencing chromatograms at polymorphic sites. Peak measurements from experimental samples were compared to a standard curve generated by mixing in vitro transcribed RNA from BYDV-PAV and PAS templates in several ratios (ranging from 1:9 to 9:1 PAV:PAS) prior to RT-PCR amplification and sequencing. The relative amount of RNA template added to a sample was regressed onto the proportion of the chromatogram peak height or area corresponding to one virus species. The function of the best fit line was used to calculate template frequency in the experimental samples.