Application of an optimized annotation pipeline to the Cryptococcus deuterogattii genome 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 anno tation 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 spe cies 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 trans porters 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 subtelo meric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.