Computational protocol: Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction

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Protocol publication

[…] Library preparation and Illumina sequencing were performed at the Ecole normale superieure genomic platform (Paris, France). Messenger (polyA+) RNAs were purified from 1 µg of total RNA using oligo(dT) primer. Libraries were prepared using the strand non-specific RNA-Seq library preparation TruSeq RNA Sample Prep Kits v1 (Illumina). Libraries were multiplexed by four on one single flowcell lane and subjected to 50-bp paired-end read sequencing on a HiSeq 1500 device. A mean of 52 ± 10 million passing Illumina quality filter reads was obtained for each of all the samples. We performed RNA-seq analysis using the Eoulsan pipeline []. Only read 1 was considered for the further analyses. Before mapping, poly N read tails were trimmed, reads ≤11 bases were removed, and reads with quality mean ≤12 were discarded. Reads were then aligned against the T. reesei genome (version 2 from the JGI website) using the Bowtie mapper (version 0.12.7) []. Alignments from reads matching more than once on the reference genome were removed using Java version of samtools []. To compute gene expression, the T. reesei genome annotation from JGI (version 2) was used. All overlapping regions between alignments and referenced exons were counted. The sample counts were normalized using DESeq 1.8.3 []. Statistical treatments and differential analyses were also performed using DESeq 1.8.3. A threshold of two for the log2 ratios with an adjusted p value <0.001 was used to identify genes differentially expressed (DE). Gene functions were identified by the aid of a manually annotated T. reesei genome database proprietary to IFPEN.The RNA-Seq gene expression data and raw fastq files are available at the GEO repository (http://www.ncbi.nlm.nih.gov.gate1.inist.fr/geo/) under Accession Number GSE89199. [...] Illumina short reads from QM9978 were mapped onto the T. reesei genome (http://genome.jgi-psf.org/Trire2/), using the Maq 0.6.6 software solution []. Mapping was done with two maximum mismatches. InDels and SNVs were identified using Maq 0.6.6. Homozygous mutations were selected and filtered on genomic context (complexity  =  1, uniqueness  > 15.8, GC percentage between 0.31 and 0.74). Each mutation was manually checked using the Integrative Genome Viewer and compared to the sequence of the Rut lineage to remove the false mutations coming from initial QM6a error sequencing. Large structural variations were searched using the BreakDancer software and filtered on the reads number covering the genomic variations [].We evaluated the location of SNVs and deletions according to gene annotations using the “filtered models” from the JGI website. From this annotation, we calculated the position of intron, promoter (using an 800 bp upstream region), and terminator (using a 200-bp downstream region).Functions of genes were predicted by similarity to orthologous genes when found in other fungal taxa using the FUNGIpath database (http://embg.igmors.u-psud.fr/fungipath/). If no orthologous gene function could be identified but a gene with a homology e-value superior to e-10 was identified, the product of this gene was annotated as “similar to.” Conserved domains were identified either by interproscan (http://www.ebi.ac.uk/interpro/search/sequence-search) or by NCBI BLASTp search (https://blast.ncbi.nlm.nih.gov/Blast.cgi).To validate the translocation between chromosomes V and VII, we amplified the region around genes encoded by the protein IDs 80028 and 54675 by PCR using oligonucleotides up and downstream of the coding region (Primer 54675_MutaC_F + 54675_MutaC_R and 80028_MutaC_F + 80028_MutaC_R Fig. b; Additional file : Table S2). PCR amplicons were sequenced with adequate primers using Sanger cycle sequencing/capillary electrophoresis (Microsynth AG, Austria). […]

Pipeline specifications

Software tools Eoulsan, Bowtie, SAMtools, DESeq, BreakDancer, InterProScan, BLASTP
Databases FUNGIpath
Applications RNA-seq analysis, Genome data visualization
Organisms Trichoderma reesei, Neurospora crassa
Chemicals Carbohydrates