Fungal RNA-seq Data Benchmarking

Three RNA-seq datasets from the industrial T. versatilis were at our disposal (unpublished data) and were prepared from: 1) growth of the mycelium on MM for 48 h (reference condition); 2) transfer of the water-rinsed mycelium to MM with ball-milled wheat straw 1% (w/v) as carbon source and sampling after 24 h; 3) direct addition of glucose at 1% final concentration to the mycelium exposed to wheat straw, and sampling after 5 h. These RNA-seq data were used for the pre-selection of stable genes (fold change (FC) equal to one, see Additional file 2) after calculating the FC as follow: RPKM (Reads Per Kilobase of exon model per Million mapped reads) value in the sample of interest / RPKM in the reference condition, for each gene. Similarly, FC for candidate reference genes were calculated from RNA-seq data publicly available at the NCBI GEO database [48 (link),49 (link)]. To identify the homologues of T. versatilis selected reference genes in the different fungi, a standard protein BLAST (blastp) using the amino-acid sequence from T. versatilis was performed against protein databases, specifying the organism. Each homologous sequence was then used for a reciprocal BLAST against the T. versatilis database in order to confirm the accuracy of the result. The detailed list of locus tags for each gene in every fungus is available in the Additional file 3. For each GOI in these studies, the ratio between the expression in a condition of interest and the expression in the control condition was calculated. Collected datasets were from Trichoderma reesei ([50 (link)], accession #GSE44648), Aspergillus niger ([51 (link)], #GSE33852), Aspergillus flavus ([52 (link),53 (link)], #GSE40202 and #GSE30031), Aspergillus fumigatus (#GSE30579), Aspergillus oryzae ([54 (link)], #GSE18851), Aspergillus nidulans ([55 (link)], #GSE44100), Blumeria graminis ([11 (link)], #GSE43163), Colletotrichum graminicola ([56 (link)], # GSE34632), Colletotrichum higginsianum ([56 (link)], #GSE33683), Fibroporia radiculosa ([57 (link)], #GSE35333), Magnaporthe oryzae ([58 (link)], #GSE30327), Neurospora crassa ([55 (link)], #GSE44100), ([59 (link)], #GSE35227), ([60 (link)], #GSE36719), Pyronema omphalodes ([61 (link)], #GSE41631), Rhizoctonia solani ([12 (link)], #GSE32577), Sordaria macrospora ([62 (link)], #GSE33668). We also accessed unpublished data from Rhizophagus irregularis ([7 (link)], #SRX375378 at NCBI Short Read Archive) and Leptosphaeria maculans (personal communication from T. Rouxel, INRA-Bioger, Thiverval-Grignon, France).