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MitoFish and MiFish Pipeline: A Mitochondrial Genome Database of Fish with an Analysis Pipeline for Environmental DNA Metabarcoding

MitoFish and MiFish Pipeline: A Mitochondrial Genome Database of Fish with an Analysis Pipeline... Fish mitochondrial genome (mitogenome) data form a fundamental basis for revealing vertebrate evolution and hydro- sphere ecology. Here, we report recent functional updates of MitoFish, which is a database of fish mitogenomes with a precise annotation pipeline MitoAnnotator. Most importantly, we describe implementation of MiFish pipeline for metabarcoding analysis of fish mitochondrial environmental DNA, which is a fast-emerging and powerful technology in fish studies. MitoFish, MitoAnnotator, and MiFish pipeline constitute a key platform for studies of fish evolution, ecology, and conservation, and are freely available at http://mitofish.aori.u-tokyo.ac.jp/ (last accessed April 7th, 2018). Key words: database, fish, mitochondrial genome, metabarcoding, environmental DNA. It has been proved that fish (and tetrapod) mitochondrial Introduction DNA can be efficiently amplified by PCR from various envi- Fish occupy an important position in the vertebrate evolution ronmental samples that include seawater, freshwater, sedi- and hydrosphere ecology, and genetic information from their ment, and gut content (Miya et al. 2015; Ushio et al. 2017). mitochondrial genomes (mitogenomes) plays a key role in eDNA analysis is a cost-effective and high-throughput ap- the investigation of their evolutionary histories and the pro- proach to investigate species diversity in a noninvasive way, tection and management of biological diversity. Mitofish is a although several factors such as potential contaminations database of fish mitogenomes with precise de novo annota- need to be taken cared of. MiFish is a set of universal PCR tions, and freely available at http://mitofish.aori.u-tokyo.ac.jp/. primers for effective metabarcoding of fish eDNA (Miya et al. Since its major update in 2013 (Iwasaki et al. 2013), MitoFish 2015). As a powerful metabarcoding tool for biodiversity has been actively and widely used by those in evolutionary monitoring, MiFish primers were developed to target a hy- science, ecology, ichthyology, fisheries, and conservation sci- pervariable region within the fish mitochondrial 12S rRNA ence from academia, government, and industry. MitoFish and gene that is flanked by two highly conservative regions based its fish mitogenome annotation pipeline MitoAnnotator now on the MitoFish data. receive >40,000 page views per year from around the world. MiFish pipeline on the MitoFish server is a user-friendly In addition to its regular update of the data content, MitoFish pipeline for analyzing fish metabarcoding data to estimate the has acquired two new functions since 2013. One is the mul- species composition and ecological characteristics of natural environment. Whereas a number of computational tools are tiple sequence annotation function, through which users can available for microbial metabarcoding analysis, there are few easily annotate many mitogenomic sequences for phylogeo- forthe eDNA metabarcodinganalysisoflargerorganisms. graphic studies, for example. MiFish pipeline serves as a useful tool for those who are in- Theother,moreimportant recent functional development terested in diversity and ecological studies of fishes. in MitoFish is the implementation of MiFish pipeline (http:// mitofish.aori.u-tokyo.ac.jp/mifish/). The recent advance in the MiFish Pipeline high-throughput sequencing technology has enabled a new powerful approach in fish studies, that is, the metabarcoding As a new function on the MitoFish server, MiFish pipeline analysis of environmental DNA (eDNA) (Deiner et al. 2017). accepts and analyzes fish mitogenomic metabarcoding data, The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is Open Access properly cited. Mol. Biol. Evol. 35(6):1553–1555 doi:10.1093/molbev/msy074 Advance Access publication April 14, 2018 1553 Downloaded from https://academic.oup.com/mbe/article/35/6/1553/4970566 by DeepDyve user on 20 July 2022 Sato et al. doi:10.1093/molbev/msy074 MBE FIG.1. Screenshots of MiFish pipeline. (A) An overview of the pipeline. (B) A view of a progress status shown during pipeline execution. (C)An HTML report that contains results of the species assignment, sequence counts, and web-page links. For each species, a detailed report with confidence scores is provided. which include those produced using the MiFish primers ðÞ aligned length of the top-hit sequence = (fig. 1A). The overall sequence quality is assessed by ðÞ mismatch numbers of the top-hit sequence þ 1 ln FastQC (http://www.bioinformatics.babraham.ac.uk/ ðÞ aligned length of the second-hit sequence = projects/fastqc/) and low-quality (Phred score< 10 by 0 ðÞ mismatch numbers of the second-hit sequence þ 1 default) 3 -tails are trimmed by DynamicTirm.pl (Cox et al. 2010). The paired-end reads are merged by FLASH (Magoc and Salzberg 2011) and erroneous merged reads All-species and within-species molecular phylogenetic that contain N-nucleotides or do not have typical lengths trees are estimated for each environmental sample. are removed (2296 25 bp by default). The primer Multiple sequence alignments are generated by MAFFT sequences are removed by TagCleaner (Schmieder et al. (Katoh and Standley 2013) and neighbor-joining phylogenetic 2010) by allowing three-base mismatches at the maxi- trees are estimated by Morphy (Adachi and Hasegawa 1992). mum. Species-level taxonomic assignment is performed An HTML report is finally presented, which can also be used using Uclust (Edgar 2010) and NCBI Blastþ (Camacho for calculating ecological indices such as alpha diversity, beta et al. 2009; fig. 1B). Redundant sequences are merged diversity, and correlation coefficients (fig. 1C). This report also into one sequence by keeping the count information. contains links to major databases such as FishBase (Froese Then, low read-number sequences (<10 by default) are and Pauly 2017), Barcode of Life (Ratnasingham and Hebert remapped onto high read-number sequences (10) at a 2007), Global Biodiversity Information Facility (Edwards given sequence-similarity threshold (99% by default), and 2004), and MitoFish. the unmapped sequences are discarded. Blastn searches Figure 2 shows an example of fish eDNA analysis results are conducted against MitoFish as a reference database produced by MiFish pipeline. The water sample was taken at with cutoff values of identity 97% and e-value 10 ,and Uchidomari river in Okinawa Island, Japan (fig. 2A)and fil- species names of the top-hit sequences are retrieved. If trated up to 960 ml using 0.45 mm Sterivex filter (Millipore) or the second to fifth top-hit sequences of each Blast search 0.70 mm glass-fiber filter (Whatman GF/F). DNA was contain those of different species, confidence scores of extracted using DNeasy PowerWater Sterivex and DNeasy the species assignment are calculated using the following Blood & Tissue kits (Qiagen) from the Sterivex and formula: 1554 Downloaded from https://academic.oup.com/mbe/article/35/6/1553/4970566 by DeepDyve user on 20 July 2022 MitoFish and MiFish Pipeline doi:10.1093/molbev/msy074 MBE A contamination and need to be interpreted with caution. Taken together, MitoFish, MitoAnnotator, and MiFish pipe- line constitute a key platform for studies of evolution, ecology, and conservation of fishes. Acknowledgments The authors thank Mutsumi Nishida, Koichiro Yamada, TakashiP.Satoh,Ryota Isagozawa, Yasunobu Maeda, Kohji Mabuchi, and Hirohiko Takeshima for their help in the devel- opment of MitoFish. This work was supported by the Japan Science and Technology Agency (CREST), the Japan Society for the Promotion of Science (Grant Numbers 15K18590, 16H06279, 17K19298, and 17HP8031), the University of the Ryukyus (the Spatiotemporal Genomics Project), and the Canon Foundation. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. Sequence data are deposited in DDBJ Sequence Read Archive with an accession number DRA006629. References Adachi J, Hasegawa M. 1992. MOLPHY: programs for molecular phylo- genetics, I. PROTML: maximum likelihood inference of protein phy- logeny. Computer Science Monographs, No. 27. Tokyo: Institute of Statistical Mathematics. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLASTþ: architecture and applications. BMC Bioinformatics 10:421. Cox MP, Peterson DA, Biggs PJ. 2010. SolexaQA: at-a-glance quality as- sessment of Illumina second-generation sequencing data. BMC Bioinformatics 11:485. FIG.2. Application of MiFish pipeline to a fish eDNA data set. (A) Deiner K, Bik HM, M€ achler E, Seymour M, Lacoursie `re-Roussel A, Photos of the sampling site in Okinawa Island, Japan. (B) A summary Altermatt F, Creer S, Bista I, Lodge DM, de Vere N, et al. 2017. of the species assignment results produced by MiFish pipeline. The Environmental DNA metabarcoding: transforming how we survey left and right bars indicate the data of eDNA extracted using the animal and plant communities. Mol Ecol. 26(21):5872–5895. Sterivex and glass-fiber filters, respectively. Read numbers are Edgar RC. 2010. Search and clustering orders of magnitude faster than expressed as percentages to the total read numbers. Nonnative spe- BLAST. Bioinformatics 26(19):2460–2461. cies in Okinawa Island are highlighted (Yellow: Invasive species, Pink: Edwards JL. 2004. Research and societal benefits of the Global Salmons). Fish pictures are from FishBase. Biodiversity Information Facility. Bioscience 54:485–486. Froese R, Pauly D. 2017. FishBase. Available from: www.fishbase.org, ver- glass-fiber filters, respectively. MiFish primers (Miya et al. sion October, 2017. Iwasaki W, Fukunaga T, Isagozawa R, Yamada K, Maeda Y, Satoh TP, 2015) were used to amplify eDNA with the annealing tem- Sado T, Mabuchi K, Takeshima H, Miya M, Nishida M. 2013. MitoFish perature of 60 C. MiSeq with V2 chemistry (Illumina) was and MitoAnnotator: a mitochondrial genome database of fish with used for 150-bp paired-end sequencing. an accurate and automatic annotation pipeline. MolBiolEvol. The estimated species composition well represented the 30(11):2531–2540. fish community in the rivers in Okinawa Island (fig. 2B). Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment soft- ware version 7: improvements in performance and usability. Mol Biol Dominant species included those typically observed in Evol. 30(4):772–780. Okinawa Island rivers such as gobies (e.g., Tridentiger kuroi- Magoc T, Salzberg S. 2011. FLASH: fast length adjustment of short reads wae, Rhinogobius giurinus,and Redigobius bikolanus), mullets to improve genome assemblies. Bioinformatics 27(21):2957–2963. (e.g., Chelon macrolepis and Chelon affinis), and giant mottled Miya M, Sato Y, Fukunaga T, Sado T, Poulsen JY, Sato K, Minamoto T, eel (Anguilla marmorata), as well as invasive alien species such Yamamoto S, Yamanaka H, Araki H, et al. 2015. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from as nonnative tilapias (genus Oreochromis) and plecos fishes: detection of more than 230 subtropical marine species. RSoc (Hypostomus plecostomus). It may be noted that salmons Open Sci. 2(7):150088. (genus Onchorhynchus) also appeared in the list presumably Ratnasingham S, Hebert PD. 2007. bold: the Barcode of Life Data System because they are widely consumed as food in Okinawa and (http://www.barcodinglife.org). Mol Ecol Notes 7(3):355–364. drainage likely contains their eDNA. These results would ex- Schmieder R, Lim YW, Rohwer F, Edwards R. 2010. TagCleaner: identi- fication and removal of tag sequences from genomic and metage- emplify that MiFish pipeline is a useful tool for eDNA analysis nomic datasets. BMC Bioinformatics 11:341. of the endemic fish species composition, invasive species, and Ushio M, Fukuda H, Inoue T, Makoto K, Kishida O, Sato K, Murata K, also impact of human activities, whereas the detection of Nikaido M, Sado T, Sato Y, et al. 2017. Environmental DNA enables salmons also suggests that eDNA analysis can be affected detection of terrestrial mammals from forest pond water. Mol Ecol by unexpected environmental influences and/or Resour. 17(6):e63–e75. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Biology and Evolution Oxford University Press

MitoFish and MiFish Pipeline: A Mitochondrial Genome Database of Fish with an Analysis Pipeline for Environmental DNA Metabarcoding

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Oxford University Press
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Copyright © 2022 Society for Molecular Biology and Evolution
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Abstract

Fish mitochondrial genome (mitogenome) data form a fundamental basis for revealing vertebrate evolution and hydro- sphere ecology. Here, we report recent functional updates of MitoFish, which is a database of fish mitogenomes with a precise annotation pipeline MitoAnnotator. Most importantly, we describe implementation of MiFish pipeline for metabarcoding analysis of fish mitochondrial environmental DNA, which is a fast-emerging and powerful technology in fish studies. MitoFish, MitoAnnotator, and MiFish pipeline constitute a key platform for studies of fish evolution, ecology, and conservation, and are freely available at http://mitofish.aori.u-tokyo.ac.jp/ (last accessed April 7th, 2018). Key words: database, fish, mitochondrial genome, metabarcoding, environmental DNA. It has been proved that fish (and tetrapod) mitochondrial Introduction DNA can be efficiently amplified by PCR from various envi- Fish occupy an important position in the vertebrate evolution ronmental samples that include seawater, freshwater, sedi- and hydrosphere ecology, and genetic information from their ment, and gut content (Miya et al. 2015; Ushio et al. 2017). mitochondrial genomes (mitogenomes) plays a key role in eDNA analysis is a cost-effective and high-throughput ap- the investigation of their evolutionary histories and the pro- proach to investigate species diversity in a noninvasive way, tection and management of biological diversity. Mitofish is a although several factors such as potential contaminations database of fish mitogenomes with precise de novo annota- need to be taken cared of. MiFish is a set of universal PCR tions, and freely available at http://mitofish.aori.u-tokyo.ac.jp/. primers for effective metabarcoding of fish eDNA (Miya et al. Since its major update in 2013 (Iwasaki et al. 2013), MitoFish 2015). As a powerful metabarcoding tool for biodiversity has been actively and widely used by those in evolutionary monitoring, MiFish primers were developed to target a hy- science, ecology, ichthyology, fisheries, and conservation sci- pervariable region within the fish mitochondrial 12S rRNA ence from academia, government, and industry. MitoFish and gene that is flanked by two highly conservative regions based its fish mitogenome annotation pipeline MitoAnnotator now on the MitoFish data. receive >40,000 page views per year from around the world. MiFish pipeline on the MitoFish server is a user-friendly In addition to its regular update of the data content, MitoFish pipeline for analyzing fish metabarcoding data to estimate the has acquired two new functions since 2013. One is the mul- species composition and ecological characteristics of natural environment. Whereas a number of computational tools are tiple sequence annotation function, through which users can available for microbial metabarcoding analysis, there are few easily annotate many mitogenomic sequences for phylogeo- forthe eDNA metabarcodinganalysisoflargerorganisms. graphic studies, for example. MiFish pipeline serves as a useful tool for those who are in- Theother,moreimportant recent functional development terested in diversity and ecological studies of fishes. in MitoFish is the implementation of MiFish pipeline (http:// mitofish.aori.u-tokyo.ac.jp/mifish/). The recent advance in the MiFish Pipeline high-throughput sequencing technology has enabled a new powerful approach in fish studies, that is, the metabarcoding As a new function on the MitoFish server, MiFish pipeline analysis of environmental DNA (eDNA) (Deiner et al. 2017). accepts and analyzes fish mitogenomic metabarcoding data, The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is Open Access properly cited. Mol. Biol. Evol. 35(6):1553–1555 doi:10.1093/molbev/msy074 Advance Access publication April 14, 2018 1553 Downloaded from https://academic.oup.com/mbe/article/35/6/1553/4970566 by DeepDyve user on 20 July 2022 Sato et al. doi:10.1093/molbev/msy074 MBE FIG.1. Screenshots of MiFish pipeline. (A) An overview of the pipeline. (B) A view of a progress status shown during pipeline execution. (C)An HTML report that contains results of the species assignment, sequence counts, and web-page links. For each species, a detailed report with confidence scores is provided. which include those produced using the MiFish primers ðÞ aligned length of the top-hit sequence = (fig. 1A). The overall sequence quality is assessed by ðÞ mismatch numbers of the top-hit sequence þ 1 ln FastQC (http://www.bioinformatics.babraham.ac.uk/ ðÞ aligned length of the second-hit sequence = projects/fastqc/) and low-quality (Phred score< 10 by 0 ðÞ mismatch numbers of the second-hit sequence þ 1 default) 3 -tails are trimmed by DynamicTirm.pl (Cox et al. 2010). The paired-end reads are merged by FLASH (Magoc and Salzberg 2011) and erroneous merged reads All-species and within-species molecular phylogenetic that contain N-nucleotides or do not have typical lengths trees are estimated for each environmental sample. are removed (2296 25 bp by default). The primer Multiple sequence alignments are generated by MAFFT sequences are removed by TagCleaner (Schmieder et al. (Katoh and Standley 2013) and neighbor-joining phylogenetic 2010) by allowing three-base mismatches at the maxi- trees are estimated by Morphy (Adachi and Hasegawa 1992). mum. Species-level taxonomic assignment is performed An HTML report is finally presented, which can also be used using Uclust (Edgar 2010) and NCBI Blastþ (Camacho for calculating ecological indices such as alpha diversity, beta et al. 2009; fig. 1B). Redundant sequences are merged diversity, and correlation coefficients (fig. 1C). This report also into one sequence by keeping the count information. contains links to major databases such as FishBase (Froese Then, low read-number sequences (<10 by default) are and Pauly 2017), Barcode of Life (Ratnasingham and Hebert remapped onto high read-number sequences (10) at a 2007), Global Biodiversity Information Facility (Edwards given sequence-similarity threshold (99% by default), and 2004), and MitoFish. the unmapped sequences are discarded. Blastn searches Figure 2 shows an example of fish eDNA analysis results are conducted against MitoFish as a reference database produced by MiFish pipeline. The water sample was taken at with cutoff values of identity 97% and e-value 10 ,and Uchidomari river in Okinawa Island, Japan (fig. 2A)and fil- species names of the top-hit sequences are retrieved. If trated up to 960 ml using 0.45 mm Sterivex filter (Millipore) or the second to fifth top-hit sequences of each Blast search 0.70 mm glass-fiber filter (Whatman GF/F). DNA was contain those of different species, confidence scores of extracted using DNeasy PowerWater Sterivex and DNeasy the species assignment are calculated using the following Blood & Tissue kits (Qiagen) from the Sterivex and formula: 1554 Downloaded from https://academic.oup.com/mbe/article/35/6/1553/4970566 by DeepDyve user on 20 July 2022 MitoFish and MiFish Pipeline doi:10.1093/molbev/msy074 MBE A contamination and need to be interpreted with caution. Taken together, MitoFish, MitoAnnotator, and MiFish pipe- line constitute a key platform for studies of evolution, ecology, and conservation of fishes. Acknowledgments The authors thank Mutsumi Nishida, Koichiro Yamada, TakashiP.Satoh,Ryota Isagozawa, Yasunobu Maeda, Kohji Mabuchi, and Hirohiko Takeshima for their help in the devel- opment of MitoFish. This work was supported by the Japan Science and Technology Agency (CREST), the Japan Society for the Promotion of Science (Grant Numbers 15K18590, 16H06279, 17K19298, and 17HP8031), the University of the Ryukyus (the Spatiotemporal Genomics Project), and the Canon Foundation. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. Sequence data are deposited in DDBJ Sequence Read Archive with an accession number DRA006629. References Adachi J, Hasegawa M. 1992. MOLPHY: programs for molecular phylo- genetics, I. PROTML: maximum likelihood inference of protein phy- logeny. Computer Science Monographs, No. 27. Tokyo: Institute of Statistical Mathematics. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLASTþ: architecture and applications. BMC Bioinformatics 10:421. Cox MP, Peterson DA, Biggs PJ. 2010. SolexaQA: at-a-glance quality as- sessment of Illumina second-generation sequencing data. BMC Bioinformatics 11:485. FIG.2. Application of MiFish pipeline to a fish eDNA data set. (A) Deiner K, Bik HM, M€ achler E, Seymour M, Lacoursie `re-Roussel A, Photos of the sampling site in Okinawa Island, Japan. (B) A summary Altermatt F, Creer S, Bista I, Lodge DM, de Vere N, et al. 2017. of the species assignment results produced by MiFish pipeline. The Environmental DNA metabarcoding: transforming how we survey left and right bars indicate the data of eDNA extracted using the animal and plant communities. Mol Ecol. 26(21):5872–5895. Sterivex and glass-fiber filters, respectively. Read numbers are Edgar RC. 2010. Search and clustering orders of magnitude faster than expressed as percentages to the total read numbers. Nonnative spe- BLAST. Bioinformatics 26(19):2460–2461. cies in Okinawa Island are highlighted (Yellow: Invasive species, Pink: Edwards JL. 2004. Research and societal benefits of the Global Salmons). Fish pictures are from FishBase. Biodiversity Information Facility. Bioscience 54:485–486. Froese R, Pauly D. 2017. FishBase. Available from: www.fishbase.org, ver- glass-fiber filters, respectively. MiFish primers (Miya et al. sion October, 2017. Iwasaki W, Fukunaga T, Isagozawa R, Yamada K, Maeda Y, Satoh TP, 2015) were used to amplify eDNA with the annealing tem- Sado T, Mabuchi K, Takeshima H, Miya M, Nishida M. 2013. MitoFish perature of 60 C. MiSeq with V2 chemistry (Illumina) was and MitoAnnotator: a mitochondrial genome database of fish with used for 150-bp paired-end sequencing. an accurate and automatic annotation pipeline. MolBiolEvol. The estimated species composition well represented the 30(11):2531–2540. fish community in the rivers in Okinawa Island (fig. 2B). Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment soft- ware version 7: improvements in performance and usability. Mol Biol Dominant species included those typically observed in Evol. 30(4):772–780. Okinawa Island rivers such as gobies (e.g., Tridentiger kuroi- Magoc T, Salzberg S. 2011. FLASH: fast length adjustment of short reads wae, Rhinogobius giurinus,and Redigobius bikolanus), mullets to improve genome assemblies. Bioinformatics 27(21):2957–2963. (e.g., Chelon macrolepis and Chelon affinis), and giant mottled Miya M, Sato Y, Fukunaga T, Sado T, Poulsen JY, Sato K, Minamoto T, eel (Anguilla marmorata), as well as invasive alien species such Yamamoto S, Yamanaka H, Araki H, et al. 2015. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from as nonnative tilapias (genus Oreochromis) and plecos fishes: detection of more than 230 subtropical marine species. RSoc (Hypostomus plecostomus). It may be noted that salmons Open Sci. 2(7):150088. (genus Onchorhynchus) also appeared in the list presumably Ratnasingham S, Hebert PD. 2007. bold: the Barcode of Life Data System because they are widely consumed as food in Okinawa and (http://www.barcodinglife.org). Mol Ecol Notes 7(3):355–364. drainage likely contains their eDNA. These results would ex- Schmieder R, Lim YW, Rohwer F, Edwards R. 2010. TagCleaner: identi- fication and removal of tag sequences from genomic and metage- emplify that MiFish pipeline is a useful tool for eDNA analysis nomic datasets. BMC Bioinformatics 11:341. of the endemic fish species composition, invasive species, and Ushio M, Fukuda H, Inoue T, Makoto K, Kishida O, Sato K, Murata K, also impact of human activities, whereas the detection of Nikaido M, Sado T, Sato Y, et al. 2017. Environmental DNA enables salmons also suggests that eDNA analysis can be affected detection of terrestrial mammals from forest pond water. Mol Ecol by unexpected environmental influences and/or Resour. 17(6):e63–e75.

Journal

Molecular Biology and Evolution – Oxford University Press

Published: Jun 1, 2018

There are no references for this article.