MetaWorks: A flexible, scalable bioinformatic pipeline for high-throughput multi-marker biodiversity assessments

HMM analysis [58]. MetaWorks aims to extend the COI metabarcode toolkit that provides a harmonized environment where data from other organismal markers in multi-marker, multi- trophic studies can also be analyzed. Conclusion MetaWorks is provided as free and open software that is versioned, can be deployed in a Conda environment, and is supported by a suite of classifiers for popular metabarcoding markers. The software comes with a small set of raw data and a step-by-step tutorial to help users gain experience quickly. There is extensive online documentation available including detailed explanations of the pipeline, available workflows, and a tutorial for new users who have never used Conda or Snakemake before. MetaWorks generates a CSV file that lists all sequence clusters, for each sample, with associated read counts, taxonomic assignments, and bootstrap support values. Numerous statistics and log files are also provided so that users can track the number of reads that pass each major bioinformatic step. Given the current use of MetaWorks by large-scale national initiatives such as STREAM and Ecobiomics, we foresee additional developments and enhancements. Future planned improvements include the devel- opment of additional HMM models for pseudogene filtering, updated and additional classifi- ers for taxonomic assignment, and support for processing larger jobs both on HPCs and in a cloud environment. We welcome suggestions and potential collaborative work to further advance this pipeline for the scientific community. Acknowledgments

We would like to thank Josip Rudar, Katie M. McGee, Chloe V. Robinson, Victoria C. Mait- land, and Michael T.G. Wright from the Hajibabaei lab for helpful discussions and testing the pipeline with various datasets. We also thank Artin Mashayekhi for assistance with the Meta- Works website. Author Contributions Conceptualization: Teresita M. Porter, Mehrdad Hajibabaei. Formal analysis: Teresita M. Porter. Funding acquisition: Mehrdad Hajibabaei. Methodology: Teresita M. Porter. Resources: Mehrdad Hajibabaei. Software: Teresita M. Porter. Supervision: Mehrdad Hajibabaei. Validation: Teresita M. Porter. Visualization: Teresita M. Porter. Writing – original draft: Teresita M. Porter. Writing – review & editing: Teresita M. Porter, Mehrdad Hajibabaei. References 1. Pace NR. A Molecular View of Microbial Diversity and the Biosphere. Science. 1997; 276: 734–740. https://doi.org/10.1126/science.276.5313.734 PMID: 9115194 PLOS ONE MetaWorks mult-marker bioinformatic pipeline PLOS ONE | https://doi.org/10.1371/journal.pone.0274260 September 29, 2022 8 / 11