Seeking Advice on Validating RNA-Seq Data Before Differential Analysis
Hello, I recently received the RNA-Seq analysis results for a set of samples from a sequencing company. My plan involves the following steps:
- Perform differential gene expression analysis to obtain a list of differentially expressed genes.
- Utilize Gene Ontology analysis to determine the functional distribution of the differentially expressed genes.
- Conduct KEGG pathway analysis to understand whether the differentially expressed genes are concentrated in specific biological pathways.
- Integrate the results from GO and KEGG analyses to identify target genes.
- Validate the findings through experimental techniques such as qPCR.
My question is, should I validate the reliability of the transcriptome data provided by the sequencing company before proceeding with these five steps? Your insights and experiences would be greatly appreciated!
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Define 'validate'. Should you do a quality control towards the sequencing data itself (fastqc) and some checks towards sample clustering and presence of batch effects? Yes, absolutely. You should always check any data before any analysis.