ABSTRACT: Nucleotide sequence and taxonomy reference databases are critical resources for widespread applications including marker-gene and metagenome sequencing for microbiome analysis, diet metabarcoding, and environmental DNA (eDNA) surveys. Reproducibly generating, managing, using, and evaluating nucleotide sequence and taxonomy reference databases creates a significant bottleneck for researchers aiming to generate custom sequence databases. Furthermore, database composition drastically influences results, and lack of standardization limits cross-study comparisons. To address these challenges, we developed RESCRIPt, a Python 3 software package and QIIME 2 plugin for reproducible generation and management of reference sequence taxonomy databases, including dedicated functions that streamline creating databases from popular sources, and functions for evaluating, comparing, and interactively exploring qualitative and quantitative characteristics across reference databases. To highlight the breadth and capabilities of RESCRIPt, we provide several examples for working with popular databases for microbiome profiling (SILVA, Greengenes, NCBI-RefSeq, GTDB), eDNA and diet metabarcoding surveys (BOLD, GenBank), as well as for genome comparison. We show that bigger is not always better, and reference databases with standardized taxonomies and those that focus on type strains have quantitative advantages, though may not be appropriate for all use cases. Most databases appear to benefit from some curation (quality filtering), though sequence clustering appears detrimental to database quality. Finally, we demonstrate the breadth and extensibility of RESCRIPt for reproducible workflows with a comparison of global hepatitis genomes. RESCRIPt provides tools to democratize the process of reference database acquisition and management, enabling researchers to reproducibly and transparently create reference materials for diverse research applications. RESCRIPt is released under a permissive BSD-3 license at https://github.com/bokulich-lab/RESCRIPt. Author summary Generating and managing sequence and taxonomy reference data presents a bottleneck to many researchers, whether they are generating custom databases or attempting to format existing, curated reference databases for use with standard sequence analysis tools. Evaluating database quality and choosing the “best” database can be an equally formidable challenge. We developed RESCRIPt to alleviate this bottleneck, supporting reproducible, streamlined generation, curation, and evaluation of reference sequence databases. RESCRIPt uses QIIME 2 artifact file formats, which store all processing steps as data provenance within each file, allowing researchers to retrace the computational steps used to generate any given file. We used RESCRIPt to benchmark several commonly used marker-gene sequence databases for 16S rRNA genes, ITS, and COI sequences, demonstrating both the utility of RESCRIPt to streamline use of these databases, but also to evaluate several qualitative and quantitative characteristics of each database. We show that larger databases are not always best, and curation steps to reduce redundancy and filter out noisy sequences may be beneficial for some applications. We anticipate that RESCRIPt will streamline the use, management, and evaluation/selection of reference database materials for microbiomics, diet metabarcoding, eDNA, and other diverse applications.
