Data corresponding to: Evaluation of sequencing and PCR-based methods for the quantification of the viral genome formula

Viruses show great diversity in their genome organisation. Multipartite
viruses package their genome segments into separate particles, most or all
of which are required to initiate infection in the host cell. The benefits
of such seemingly inefficient genome organization are not well understood.
One hypothesised benefit of multipartition is that it allows for flexible
changes in gene expression by altering the frequency of each genome
segment in different environments, such as encountering different host
species. The ratio of the frequency of  segments is termed the
genome formula (GF). Thus far, formal studies quantifying the GF have been
performed for well-characterised virus-host systems in experimental
settings using RT-qPCR. However, to understand GF variation in natural
populations or novel virus-host systems, a comparison of several methods
for GF estimation including high-throughput sequencing (HTS) based methods
is needed. Currently, it is unclear how HTS-methods compare a golden
standard, such as RT-qPCR. Here we show a comparison of multiple GF
quantification methods (RT-qPCR, RT-digital PCR, Illumina RNAseq and
Nanopore direct RNA sequencing) using three host plants (Nicotiana
tabacum, Nicotiana benthamiana, and Chenopodium quinoa) infected with
cucumber mosaic virus (CMV), a tripartite RNA virus. Our results show that
all methods give roughly similar results, though there is a significant
method effect on genome formula estimates. While the RT-qPCR and RT-dPCR
GF estimates are congruent, the GF estimates from HTS methods deviate from
those found with PCR. Our findings emphasise the need to tailor the GF
quantification method to the experimental aim, and highlight that it may
not be possible to compare HTS and PCR-based methods directly. The
difference in results between PCR-based methods and HTS highlights that
the choice of quantification technique is not trivial.