Computational protocol: Using Network Extracted Ontologies to Identify Novel Genes with Roles in Appressorium Development in the Rice Blast Fungus Magnaporthe oryzae

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Protocol publication

[…] Gene Ontology (GO) terms [] were assigned to M. oryzae sequences using Blast2GO [] and provided by Darren Soanes []. Blast2GO has previously been used to annotate M. oryzae genes []. Briefly, M. oryzae sequences were annotated via sequence similarity using BLAST against the National Center for Biotechnology Information’s (NCBI) non redundant (NR) database with Blast2GO’s default parameters (E-value cutoff 1.0 × 10−3). Next, sequences were searched against the InterPro database, using default parameters, to associate sequences with protein families and include GO term annotations based on protein motifs and domains. Finally, the M. oryzae sequences were mapped and annotated with GO terms using default parameters, including an E-value cutoff of 1.0 × 10−6 and applying greater weight to experimentally validated GO term annotations. [...] An index for the M. oryzae genome was built using bowtie2-build and reads from RNA-Seq datasets were aligned to the reference genome using bowtie2 (version 2.2.5) []. For both single- and paired-end data, reads were aligned in the “local” alignment mode with the “very-sensitive” preset. Alignment files were post-processed (sorted, converted to binary format) using SAMtools []. The number of reads mapping to known genes was estimated using featureCounts (version 1.5.0) in an unstranded manner and using the exon feature in the M. oryzae annotation []. Expression of genes was estimated by reads per kilobase of transcript per million mapped reads (RPKM) using edgeR (version 3.16.3) with filtering to remove genes with <2 counts per million in >10% of samples []. […]

Pipeline specifications

Software tools Bowtie, SAMtools, Subread, edgeR
Application RNA-seq analysis
Organisms Magnaporthe oryzae, Oryza sativa
Diseases Mitochondrial Diseases