Computational protocol: RNA-Seq-Mediated Transcriptome Analysis of a Fiberless Mutant Cotton and Its Possible Origin Based on SNP Markers

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Protocol publication

[…] After adaptor trimming and removal of low-quality reads, RNA-seq reads were stringently aligned against the Upland cotton (AADD) TM-1 genome using the Burrows-Wheeler Alignment tool [, ]. SNPs/InDels were called using the Genome Analysis Toolkit [] based on three criteria: read depth ≥ 30, mapping quality ≥ 20, non-ambiguous reference base and the length of InDel ≥ 2. [...] Gene expression levels were measured by RNA-seq quantification analysis using the Noiseq package []. The following criteria were used to identify DEGs: false discovery rate ≤ 0.001, |log2 ratio| ≥ 1 and probability ≥ 0.85.The Blast2GO program, with a cutoff value of 1 × 10−5, was used to obtain DEG Gene Ontology (GO) annotations []. This analysis mapped all DEGs to GO terms in the database and counted the number of DEGs associated with each term. The WEGO program was then applied to plot GO functional classifications of the DEGs []. […]

Pipeline specifications

Software tools GATK, NOISeq, Blast2GO, WEGO
Application RNA-seq analysis
Organisms Gossypium hirsutum, Gossypium barbadense