2. Abstract Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterize the complete ‘infectome’ (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing (“metatranscriptomics”) we documented the presence of contaminant viral sequences in multiple libraries of ‘blank’ negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank libraries, as well as contaminating sequences from the RNA virus families Totiviridae, Tombusviridae and Lentiviridae . These data suggest that the contamination of common laboratory reagents is likely widespread and can comprise a wide variety of viruses. 3. Data summary The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. 1.5 Repositories The viral genome sequence data generated in this study has been deposited in the NCBI database under accession numbers MZ824225 - MZ824237 . Sequence reads are available at the public Sequence Read Archive (SRA) database with accession SRX6803604 and under the BioProject accession PRJNA735051 reference numbers SRR14737466-71 and BioSample numbers SAMN20355437-40.

Metagenomic identification of viral sequences in laboratory reagents