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

RESEARCH ARTICLE MetaWorks: A flexible, scalable bioinformatic pipeline for high-throughput multi-marker biodiversity assessments Teresita M. PorterID*, Mehrdad Hajibabaei Centre for Biodiversity Genomics @ Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, Guelph, ON, Canada * terrimporter@gmail.com Abstract Multi-marker metabarcoding is increasingly being used to generate biodiversity information across different domains of life from microbes to fungi to animals such as for molecular ecol- ogy and biomonitoring applications in different sectors from academic research to regulatory agencies and industry. Current popular bioinformatic pipelines support microbial and fungal marker analysis, while ad hoc methods are often used to process animal metabarcode markers from the same study. MetaWorks provides a harmonized processing environment, pipeline, and taxonomic assignment approach for demultiplexed Illumina reads for all biota using a wide range of metabarcoding markers such as 16S, ITS, and COI. A Conda environ- ment is provided to quickly gather most of the programs and dependencies for the pipeline. Several workflows are provided such as: taxonomically assigning exact sequence variants, provides an option to generate operational taxonomic units, and facilitates single-read pro- cessing. Pipelines are automated using Snakemake to minimize user intervention and facili- tate scalability. All pipelines use the RDP classifier to provide taxonomic assignments with

confidence measures. We extend the functionality of the RDP classifier for taxonomically assigning 16S (bacteria), ITS (fungi), and 28S (fungi), to also support COI (eukaryotes), rbcL (eukaryotes, land plants, diatoms), 12S (fish, vertebrates), 18S (eukaryotes, diatoms) and ITS (fungi, plants). MetaWorks properly handles ITS by trimming flanking conserved rRNA gene regions as well as protein coding genes by providing two options for removing obvious pseudogenes. MetaWorks can be downloaded from https://github.com/ terrimporter/MetaWorks and quickstart instructions, pipeline details, and a tutorial for new users can be found at https://terrimporter.github.io/MetaWorksSite. Introduction Marker gene sequencing, metabarcoding, or metasystematics are different terms for the same technique that involves extracting DNA from bulk samples such as soil, water, or mixtures of indi- viduals collected from traps. One key strength of this technique is not having to isolate or identify individual specimens. A signature DNA region is then enriched, for example using PCR, to iden- tify biological community composition using bioinformatics [1–3]. In microbial ecology to animal PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0274260 September 29, 2022 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Porter TM, Hajibabaei M (2022) MetaWorks: A flexible, scalable bioinformatic pipeline for high-throughput multi-marker biodiversity assessments. PLoS ONE 17(9): e0274260. https://doi.org/10.1371/journal. pone.0274260

Editor: Hideyuki Doi, University of Hyogo, JAPAN Received: April 22, 2022 Accepted: August 24, 2022 Published: September 29, 2022 Copyright: © 2022 Porter, Hajibabaei. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: MetaWorks runs at the command-line in a Conda environment on linux-64. Miniconda can be downloaded from https://repo.continuum.io/miniconda/Miniconda3- latest-Linux-x86_64.sh. If pseudogene filtering will be performed ORFfinder can be downloaded from the NCBI ftp site at ftp://ftp.ncbi.nlm.nih.gov/ genomes/TOOLS/ORFfinder/linux-i64/ORFfinder. gz. MetaWorks can be downloaded from GitHub at https://github.com/terrimporter/MetaWorks. Quickstart examples, detailed pipeline descriptions, a tutorial for new users, and FAQs are available from the MetaWorks website at https://