MitoFish, MitoAnnotator, and MiFish Pipeline: Updates in 10 Years

Abstract

MitoFish, MitoAnnotator, and MiFish Pipeline are a comprehensive database of fish mitochondrial genomes (mitogenomes), accurate annotation software of fish mitogenomes, and a web platform for metabarcoding analysis of fish mitochondrial environmental DNA (eDNA), respectively. The MitoFish Suite currently receives over 48,000 visits worldwide each year. Though, the performance and usefulness of the online platforms can still be improved. Here, we present essential updates on these platforms, including enrichment of the reference datasets, enhanced searching function, substantially faster genome annotation and eDNA analysis with the denoising of sequencing errors, and a multi-sample comparative analysis function. These updates have made our platform more intuitive, effective, and reliable. These updated platforms are freely available at http://mitofish.aori.u-tokyo.ac.jp/.

MultiBarcodeTools: Easy selection of optimal primers for eDNA multi‐metabarcoding

“…The remaining 34 primers target fish mitochondrial 12S rRNA, 16S rRNA, cox1 , or cytb genes (Table 1). We used the CRABS v0.1.3 program (Jeunen et al., 2022) to construct a comprehensive sequence dataset of amplicon regions of the 34 primers based on 3482 complete mitochondrial genomes (mitogenomes) from the MitoFish database (Iwasaki et al., 2013; Sato et al., 2018; Zhu et al., 2023) (v3.86, accessed on February 20, 2023) (Text S1). The dataset was stored in a MySQL database, which functions as the primary data resource for MultiBarcodeSelector.…”

“…We prepared reference mitogenomic sequences of inhabitant species in this pond based on Zenodo (https://doi.org/10.5281/zenodo.3730934) and analyzed them with the MultiBarcodePipeline and the two primers. Then, the stand‐alone MiFish pipeline (Zhu et al., 2023) was employed to identify species composition in each sample with both primers using an identity threshold of 98%, and the identification results were compared between the two primers.…”

“…For amplicon sequences with low identification confidence values, we divided 1%, 20%, or 50% of the amplicons to the second‐hit species to simulate different situations, generating three distinct simulated datasets. Finally, we used the stand‐alone MiFish pipeline to analyze the simulated reads of the second primer (Zhu et al., 2023) with an identity threshold set to 98%.…”

“…A fundamental concern in eDNA metabarcoding is that, even in a single experiment, a single metabarcoding primer set is usually insufficient and can seldom amplify and discriminate all potential inhabitant species. For example, a highly utilized fish metabarcoding primer, MiFish‐U, failed to identify approximately half of the species with high confidence in the Uchidomari River on Okinawa Island, Japan, because of the existence of closely related species that have very similar sequences in the amplified region (Zhu et al., 2023). It is also common that, despite primers being expected to bind to their target regions in silico, amplification efficiencies can become low for specific species‐primer pairs for unknown reasons, which may include atypical DNA structures or modifications at the binding site.…”

rCRUX: A rapid and versatile tool for generating metabarcoding reference libraries in R

“…By far, the most successful and widely used reference databases (e.g. Silva, PR2, UNITE, and MitoFish) rely on dedicated staff and resources to maintain and update such repositories (Guillou et al., 2012; Kõljalg et al., 2005; Quast et al., 2012; Zhu et al., 2023). Given the extensive resources needed to curate and maintain such repositories, there are only a handful of such efforts representing only commonly used loci.…”