When DNA gets in the way: A cautionary note for DNA contamination in extracellular RNA-seq studies
Jasper Verwilt, Wim Trypsteen, Ruben Van Paemel, Katleen De Preter, María D. Giraldez, Pieter Mestdagh, Jo Vandesompele
Abstract
With great interest, we read the paper by Zhou et al. (1) describing a methodology that enables extracellular RNA sequencing (exRNA-seq) from extremely low input (Small Input Liquid Volume Extracellular RNA Sequencing [SILVER-seq]). We were intrigued by the high number of detected genes compared to our previous studies (2, 3) and noticed low reproducibility. We hypothesized that these observations could originate from substantial DNA contamination. Therefore, we reanalyzed the SILVER-seq data (4) to determine the extent of DNA signal in the sequencing reads. First, we analyzed the fraction of reads mapping to the different genomic regions. We noticed that these fractions closely resembled the distributions in the genome (Fig. 1 A ). Specifically, fewer than 5% of the reads mapped to exonic regions, while our own exRNA-seq data (3) showed an average of 35% exonic reads. Secondly, we analyzed reads mapping to spliced sequences, expecting them to be … [↵][1]1To whom correspondence may be addressed. Email: Jasper.verwilt{at}ugent.be. [1]: #xref-corresp-1-1