Fig. 1

Genomic DNA contamination in RNA-seq raises concerns about the reliability of RNA-seq results. Diagram showing that genomic DNA (gDNA) contamination may affect RNA-seq results. Most RNA-seq studies focus on mRNA and/or noncoding RNA in cells or tissues, while these RNAs account for a small part of total cellular RNA. When enriching for such RNAs, gDNA will be enriched as well and eventually contaminate RNA-seq data. In extracted total RNA, the sample consists of a large amount of rRNA, small amounts of mRNAs and noncoding RNAs, and a small amount of gDNA. During library preparation, particularly using the ribosomal depletion method, most rRNA in the total RNA sample is removed, which results in high enrichment of mRNA and noncoding RNAs together with gDNA. These gDNAs will contaminate RNA-seq data and ultimately affect analyzing results, such as falsely increasing gene expression levels that may influence the DEG detection. When detecting DEGs between Treatment and Control groups, there are roughly four situations for one specific gene. Situations 1: both the Treatment and Control are not contaminated by gDNA; Situation 2: only Control is contaminated by gDNA contamination; Situation 3: only Treatment is contaminated by gDNA; Situation 4: both Treatment and Control are contaminated by gDNA. Different contaminating situations would result in different DEG detecting results for genes, e.g. gene A