RNAseq analysis of wildtype and knockout mice
Analysis:
All RNA seq FastQ files are quality assessed with FastQC. The quality score was noted for each file. Each sequences passed the quality score of basic statistics, per base sequence quality (25%-75% with a median of at least 25),per sequence quality score (above 27 indicates less than 2% error rate,) and per base sequence content (less than 10%) and GC content (difference less than 5% than mean GC content). After quality assessment all files are quality and adaptor trimmed with TRIMMOMATIC. TRIMMOMATIC output files were again assessed with FastQC to check that it maintained those quality after trimming adaptors.
Each sample sequences (two) were aligned to a single output file using RNA STAR with the mouse reference genome GrcM38.99.
Differential expression counts were done for each sample using HTseq-count. After counting all gene expressions, differential gene expression was compared using DESEQ2 between biological replicates of Wild Type (pooled together) and KnockOut (pooled together) samples. Differential expression of genes is presented in exel files as significantly differentially expressed genes between two groups. DESEQ2 results also generates MA-plot, PCA plot and other global visual files representing differential gene expression.
DESEQ2 Differentially expressed genes with significant p-value adjusted genes for log2FC (fold change) were used for volcano plot. Volcano plot with top 20 significant genes were presented with NCBI gene notation.
Heatmap of top 20 significant genes were done using normalized counts from the output of LIMMA-VOOM software, analogous to DESEQ2.
Gene ontogeny (GO) were analyzed with GOSEQ with significantly adjusted p-value and log2FC genes.
Pathway mapping were done using GOSEQ with the ID of KEGG pathway. KEGG pathway mapping was obtained using PATHVIEW.