Since the pioneering Global Ocean Sampling project, large-scale sequencing of environmental DNA has become a common approach to assess the biodiversity of diverse environments, with an emphasis on microbial populations: unicellular eukaryotes ("protists"), bacteria, archaea, and their innumerous associated viruses and phages. However, the global analysis of the viral diversity ("the virome") from sequence data is fundamentally hampered by the lack of a universal gene that would allow their unambiguous identification and reliable separation from cellular microorganisms. The problem has been made even more difficult with the discovery of micron-sized giant viruses for which the usual fractionation protocol on a "sterilizing" filter is no longer an option. In the present proof-of-principle work we used actual metagenomic data to show that glutamine-hydrolysing asparagine synthase is a reliable sequence probe to discover new members of the Mimiviridae family, hint at the existence of a new family of large DNA viruses, and point out misidentified database entries.
Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of the genus Chlorovirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.
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