Computational protocol: Analysis of genes that are differentially expressed during the Sclerotinia sclerotiorum–Phaseolus vulgaris interaction

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Protocol publication

[…] The sequences were pre-processed using Phred () and Cross Match software. Sequences with at least 100 nucleotides and a Phred quality greater than or equal to 20 were considered for further analysis. Cleaned ESTs were assembled into contigs using the CAP3 assembly program and compared against the GenBank non-redundant (nr) database using the BLASTx algorithm available at the National Center for Biotechnology Information. Functional annotation using Gene Ontology terms was analyzed with the Blast2GO program (). KEGG pathway annotation was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.Primers were designed using Software Primer Express (Applied Biosystems, Carlsbad, CA, USA) for the amplification of gene fragments that were approximately 80–150 bp in length and with an annealing temperature of 60°C (Table ). The primer specificity was checked in silico against the NCBI database2 through the Primer-BLAST tool. The selected primers were tested with PCR, and their specificity was checked by nucleotide sequencing on an ABI 3130 sequencer (Applied Biosystems, Carlsbad, CA, USA) using DyeTerminator chemistry 4 to confirm their identities. […]

Pipeline specifications

Software tools BLASTX, Blast2GO, Primer Express, Primer-BLAST
Databases KEGG PATHWAY
Application qPCR
Organisms Sclerotinia sclerotiorum, Phaseolus vulgaris, Bovine orthopneumovirus, Proteus vulgaris