TABLE 1.

Recommendation for reducing contamination in viral metagenomic studies.

	Recommendations
General practices	• Use sterile laboratory equipment: tubes, tips with filter, decontaminated racks, and machines • Wear disposable protective coats, gloves, and face masks • Always decontaminate working area • Perform wet-laboratory work under laminar flow hood • Perform all steps in dedicated laboratory areas: create separate preamplification, amplification, and postamplification sites • Minimize the number of investigators in a project and record which samples were handled by a given technician
Sampling	• Avoid cross-contamination during sample preparation • Be aware that caging multiple laboratory animals in the same space may influence their microbial composition • Collect samples in sterile tubes • Avoid contamination derived from the skin or breath of the investigator • Use rich-biomass samples Maximize the sample volume for extraction when using low-biomass material
Reagents and wet-laboratory procedures	• Use the same types of reagents during the whole project Record all batches and lot numbers of all reagents used in a project • Minimize the number of steps in wet-laboratory workflow • Use dedicated extraction kits for low-biomass samples with low elution volumes • Keep in mind that silica column–based nucleic acid extraction kits are associated with numerous contaminants • Use highly purified enzymes and polymerases with high fidelity • Minimize the number of PCR cycles during amplification • Avoid using multichannel pipettes, sample plates, and strips without separate caps • If necessary make gaps in plates between samples • Use VLP enrichment workflows • Analyze the same biological samples in repeats
Sequencing	• Sequence all samples in a given project in the same sequencing center • Use unique dual barcoding • Sequence samples with similar viral titters in the same run • Minimize the number of PCR cycles during indexing
Controls	• Use blank and negative controls during sample preparation and extraction • Use non-template controls if amplification step is included • Use a variety of positive-control titrations to verify the accuracy of metagenomic workflow
Data analysis	• Create a list of contaminants specific for your viral metagenomic workflow and laboratory • Set your own threshold for contamination detection based on your results and experience • Align NGS reads to host and bacterial genomes to examine potential contamination • Set criteria for viral detection that include matching different regions of the viral genome with sufficient genome coverage • Align contigs rather than single NGS reads to viral genomes • Check the complexity of identified viral sequences to distinguish true signals from artifacts • Take into consideration sequencing error • Use verified and filtered viral databases for viral classification • Remove PhiX phage sequences before data upload • Use open-source decontamination software • Use dedicated software for viral detection and phage identification
Data interpretation and good practices	• For clinical diagnostic application, verify all potentially causative viral agents found in SM studies using PCRs • Pay close attention and be critical with regards to non-vertebrae viruses found in virome of vertebrae hosts • Perform batch/study/investigator associations with contaminants found in your data