Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI’s RNA-seq archive

Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Because they are small, they can escape filtration in culture media. Because they lack cell walls, they are resistant to commonly used antibiotics. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series (or projects) in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as [≥] 100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. Interestingly, series using poly(A)-selection, which should bias against mycoplasma detection, had comparable contamination rates as non-poly(A)-selected series. We also examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. Lastly, to estimate the potential economic cost of this widespread contamination, we queried NIH RePORTER to find grants with the terms \"cell culture\" or \"cell lines\". Funding for these totaled over $3 billion, suggesting hundreds of millions of dollars in research are potentially affected. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality, with considerable financial consequences.