Computational protocol: Characterization of Copy Number Variation’s Potential Role in Marek’s Disease

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Protocol publication

[…] To study genes overlapping with CNVR, gene ontology categories were analyzed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system (http://www.pantherdb.org/) as described previously []. The p-values were calculated according to the binomial test integrated in the PANTHER online tool. Bonferroni’s correction for multiple testing was applied, followed by assessment of the GO significance at a p-value cutoff level of 0.05. In addition, genes overlapping with CNVR were further analyzed using Ingenuity Pathways Analysis (IPA) v9.0 (Ingenuity Systems, Redwood City, CA, USA). The accessions of unique genes were imported into the software and subsequently mapped to their corresponding annotations in the Ingenuity Pathways Knowledge Base. The “Core Analysis” function included in IPA (http://www.ingenuity.com/webcite) was utilized to explore the context of networks, biological functions, and pathways. We used only human, rat, and mouse genes and all confidence levels, including experimentally observed evidence, predicted high or moderate confidence. The top significant biological functions and pathways were considered in our study. [...] Total RNA of spleen and thymus from Line 63 and Line 72 was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and mRNA isolation was performed from total RNA using Oligotex mRNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacture’s instruction. Approximately 300 ng of mRNA was used to synthesize the first and second strands of cDNA by SuperScriptTM III Reverse Transcriptase (Invitrogen, Waltham, MA, USA) and Second Strand cDNA synthesis kits (NEB, Ipswich, MA, USA). After purification, double-strand cDNA (dscDNA) was sonicated by a Bioruptor Sonicator (Diagenode, Denville, NJ, USA). Two biological replicates were used in the RNA-Seq library construction and RNA sequencing was conducted on an Illumina Hiseq 2000 (Illumina, San Diego, CA, USA) using 50 bp single end reads (SE50). Raw sequenced data were checked for quality considering the read counts, overall read quality, and read distribution. Then we aligned clean reads onto Gallus_gallus-4.0 using Bowtie version 1.0.0 []. In order to control the mapping quality, the first 15 bps from the original 50 bp of reads were trimmed. The counting of reads per gene was executed by the summarizeOverlaps function implemented in R v3.0.2. After dispersion estimation, we used the R package edgeR and corresponding complementary functions GLM to analyze the count reads data and perform a comparison analysis. Differentially-expressed genes (DEG) were filtered by a threshold of 0.05 after FDR adjustment []. […]

Pipeline specifications

Software tools PANTHER, IPA
Application Protein sequence analysis
Organisms Gallus gallus, Cucumber necrosis virus