Gauge your phage: benchmarking of bacteriophage identification tools in metagenomic sequencing data

Background The prediction of bacteriophage sequences in metagenomic datasets has become a topic of considerable interest, leading to the development of many novel bioinformatic tools. A comparative analysis of ten state-of-the-art phage identification tools was performed to inform their usage in microbiome research. Methods Artificial contigs generated from complete RefSeq genomes representing phages, plasmids, and chromosomes, and a previously sequenced mock community containing four phage species, were used to evaluate the precision, recall, and F1 scores of the tools. We also generated a dataset of randomly shuffled sequences to quantify false-positive calls. In addition, a set of previously simulated viromes was used to assess diversity bias in each tool’s output. Results VIBRANT and VirSorter2 achieved the highest F1 scores (0.93) in the RefSeq artificial contigs dataset, with several other tools also performing well. Kraken2 had the highest F1 score (0.86) in the mock community benchmark by a large margin (0.3 higher than DeepVirFinder in second place), mainly due to its high precision (0.96). Generally, k-mer-based tools performed better than reference similarity tools and gene-based methods. Several tools, most notably PPR-Meta, called a high number of false positives in the randomly shuffled sequences. When analysing the diversity of the genomes that each tool predicted from a virome set, most tools produced a viral genome set that had similar alpha- and beta-diversity patterns to the original population, with Seeker being a notable exception. Conclusions This study provides key metrics used to assess performance of phage detection tools, offers a framework for further comparison of additional viral discovery tools, and discusses optimal strategies for using these tools. We highlight that the choice of tool for identification of phages in metagenomic datasets, as well as their parameters, can bias the results and provide pointers for different use case scenarios. We have also made our benchmarking dataset available for download in order to facilitate future comparisons of phage identification tools.