RNA-Sequencing of PEG-stressed Sesame roots in drought sensitive and tolerant genotypes

Purpose: The goals of this study are to understand transcriptional changes in the roots of drought-tolerant and sensitive sesame genotypes using PEG (Polyethylene glycol) induced osmotic stress. Overall design: Methods:The removal of sequence adapters, mapping to reference genome, and normalization of gene expression was performed using CLC Genomics Workbench (V4.5, CLC Bio.). The RNAseq reads were first assessed for quality with FastQC; adapters were removed with Cutadapt (Martin 2011) and trimmed the low-quality bases (q < 20) with FastX-toolkit (Gordon and Hannon 2010). Quality-filtered reads were then mapped to the reference Sinbase database (Wang et al. 2014) using TopHat2 (Kim et al. 2013) with default parameters. Novel transcripts were assembled by Cufflink (Trapnell et al. 2010), then combined with available gene annotation using CuffMerge. Normalized expression levels of genes were expressed in FPKM values as generated by Cuffquant and Cuffnorm running with the option “–frag-bias-correct” and “–multi-read-correct” (Trapnell et al. 2012). The differential expressed genes (DEGs) of pooled samples from each condition was determined using Cuffdiff, using the FPKM values.