Whole genome RNA sequencing of wild type and nath-10(icb99 and icb102) alleles

Using next generation RNA sequencing we aimed to investigate the role of a single point mutation in the nath-10 locus as the causative mutation driving phenotypic variability in the final number of the stem cell like seam cells in epithelium of C. elegans. To this end we performed whole genome RNA sequencing using the illumina Hiseq platform on wildtype and two nath-10 loss of function alleles, nath-10(icb99 and icb102) at the 2nd larval stage of development, around 27 hours post hatching. The sequence reads, generated by BGI (China), that passed quality controls were aligned to the most recent C. elegans (WBcel235) transcriptome using the pseudoalignment tool Kallisto. The generated abundance files were analysed using an R pipeline for DEseq2. Results showed that ~3% of the genes in nath-10 loss-of-function mutants had a significant differential change in expression, adjusted p-value <0.1 and fold change ≥1.2. We further found, on calculation of transcript coefficient of variation, global variance in transcript number across replicates was significantly increased in nath-10 loss-of-function alleles compared to wild-type. On further analysis genes that play an essential role in seam cell development were found to be increased in transcript variance upon loss of function, including the key Wnt target gene egl-18. Moreover, when analysed using single molecule fluorescence and transcriptional reporter constructs egl-18 was confirmed to more variable in the loss-of-function allele nath-10(icb102).