Application of an optimized annotation pipeline to theCryptococcus deuterogattiigenome reveals dynamic primary metabolic gene clusters and genomic impact of RNAi loss

Abstract

Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary-based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results show that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5–10 million reads per RNA-seq replicate. We also showed that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, we observed that antisense transcription was not higher in C. deuterogattii than in the two other Cryptococcus species. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

Epigenetic dynamics of centromeres and neocentromeres in Cryptococcus deuterogattii

“…deuterogattii R265 reference genome, and 5mC methylation was thought to be entirely absent based on PCR-based assays with methylation-sensitive restriction enzymes [ 21 ]. An updated gene annotation of the R265 reference genome and re-mapping of RNA-seq data revealed that the DMT5 gene was previously mis-annotated and it could encode a putative fully functional protein ( S2 Fig ) [ 41 ]. To analyze the function of 5mC DNA methylation, the DMT5 gene was deleted by homologous recombination and bisulfite sequencing was performed with DNA isolated from the wild-type strain and a dmt5 Δ deletion mutant ( S3A Fig ).…”

“…The new genome annotation shows that the DMT5 gene is full-length and has a similar length to the C . neoformans ortholog [ 41 ]. (B) Detailed view of C .…”

Cryptococcus extracellular vesicles properties and their use as vaccine platforms

“…The following databases were used. For C. neoformans KN99α, C. deneoformans JEC21 and C. deuterogattii R265 we used the recently updated proteomes (Gröhs Ferrareze et al., 2021 ; Wallace et al., 2020 ). The following search parameters were applied: carbamidomethylation of cysteines was set as a fixed modification, oxidation of methionine and protein N‐terminal acetylation were set as variable modifications.…”

“…In the present study, we used the recently described protocol (Reis et al., 2019 ) to obtain EV‐enriched samples from Cryptococcus , together with cutting edge methodological approaches to revisit Cryptococcus EV structure, cargo, and their biological functions. Here we report a detailed analysis of three species ( C. neoformans , C. deneoformans and C. deuterogattii ) for which both a good genome annotation and RNA‐Seq data were available (Gonzalez‐Hilarion et al., 2016 ; Janbon et al., 2014 ; Gröhs Ferrareze et al., 2021 ). We produced a robust set of data containing cryo‐electron microscopy (cryo‐EM) and cryo‐electron tomography (cryo‐ET) proteomics, and nanoscale flow cytometry analysis, suggesting a new EV structural model, in addition to a list of cryptococcal EV protein markers.…”

Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

“…Interestingly, the outbreak species C. deuterogattii is RNAi deficient because many genes encoding RNAi components are severely truncated or absent entirely 32 . This loss of RNAi has been shown to be associated with loss of all functional transposable elements, consequently shorter centromeres, and higher rates of intron retention 28,38 .…”