# MIxS-SA: a MIxS extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms

Fátima Jorge, Jaelle Brealey, Paul Brindley, Marie Buysse, Cinzia Cantacessi, Olivier Duron, Raina Fichorova, Connor Fitzpatrick, Megan Hahn, Christopher Hunter, et al.

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Fátima Jorge, Jaelle Brealey, Paul Brindley, Marie Buysse, Cinzia Cantacessi, et al., MIxS-SA: a MIxS extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms. ISME Communications, 2022, 2 (1), pp.9. 10.1038/s43705-022-00092-w. hal-03701954

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MIxS-SA: a MIxS extension defining the minimum
information standard for sequence data from
symbiont-associated micro-organisms

Fátima Jorge, Jaelle Brealey, Paul Brindley, Marie Buysse, Cinzia Cantacessi,
Olivier Duron, Raina Fichorova, Connor Fitzpatrick, Megan Hahn,
Christopher Hunter, et al.

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To cite this version:

Fátima Jorge, Jaelle Brealey, Paul Brindley, Marie Buysse, Cinzia Cantacessi, et al.. MIxS-SA: a MIxS extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms. ISME Communications, 2022, 2 (1), pp.9. 10.1038/s43705-022-00092-w. hal-03796265

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# MlxC-SA: a MlxC extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms

Fátima Jorge<fnref n="1" />,*, Jaelle C. Brealey², Paul J. Brindley³, Marie Buysse⁴,⁵, Cinzia Cantacessi⁶, Olivier Duron⁴,⁵, Raina Fichorova⁷, Connor R. Fitzpatrick⁸, Megan Hahn⁹, Christopher Hunter¹⁰, Vincent Hervé¹¹, Laura J. Knoll¹², Kevin D. Kohl¹³, Marco Lalle¹⁴, Julius Lukeš¹⁵, Joaquín Martínez Martínez¹⁶, Susan L. Perkins¹⁷, Robert Poulin<fnref n="1" />, Karyna Rosario¹⁸, Adam C. Schneider¹⁹, Lynn M. Schriml²⁰, Luke R. Thompson²¹,²², Ramona L. Walls²³ and Nolwenn M. Dheilly²⁴,²⁵✉

© The Author(s) 2022

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MlxC) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MlxC standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MlxC-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

ISME Communications; https://doi.org/10.1038/s43705-022-00092-w

## INTRODUCTION

Interspecific interactions are ubiquitous across the Tree of Life. With the realization that eukaryotic organisms can harbor rich microbial communities, came also the view that these smaller partners may in fact play important roles in mediating host-symbiont associations, thus adding a further layer to this complex set of nested interactions, i.e. host-symbiont-microbe [1-8]. As the number of studies exploring the microorganisms associated with symbiotic organisms increases, likewise does the need for compliant standardized metadata that provides contextual information associated with each study and sample. Standardized

metadata allows for the integration of data across organisms, resources, and within data repositories. Here, we present the symbiont-associated (SA) environmental package as a new extension to the minimum information about any (x) sequence (MlxC) standards [9], which will be included in MlxC version 6. Whilst the MlxC-SA expands upon the MlxC host-associated environmental package [9], it reflects the need for a new standard that takes into account the distinct life history traits of symbionts, their association with one or multiple host organisms, the complex nature of host-symbiont interactions along the mutualism-parasitism continuum, and the nestedness of symbiont-associated

<footnote n="1">¹Department of Zoology, University of Otago, Dunedin, New Zealand. ²Department of Natural History, NTNU University Museum, Norwegian University of Science and Technology, Trondheim, Norway. ³Department of Microbiology, Immunology and Tropical Medicine, School of Medicine & Health Sciences, George Washington University, Washington, D.C 20037, USA. ⁴MIVEGEC (Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle), Centre National de la Recherche Scientifique (CNRS)—Institut pour la Recherche et le Développement (IRD) - Université de Montpellier (UM), Montpellier, France. ⁵CREEs (Centre de Recherche en Écologie et Évolution de la Santé), Montpellier, France. ⁶Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK. ⁷Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. ⁸Biology Department, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. ⁹New York City Department of Health and Mental Hygiene, Long Island City, NY, USA. ¹⁰GigaDB Director, GigaScience Press, BGI-Hong Kong, Hong Kong, China. ¹¹Institut de Recherche sur la Biologie de l'Insecte, UMR 7261, CNRS—Université de Tours, Avenue Monge, Parc Grandmont, 37200 Tours, France. ¹²Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA. ¹³Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. ¹⁴Department of Infectious Diseases, Foodborne and Neglected Parasitic Diseases; European Union Reference Laboratory for Parasite, Istituto Superiore di Sanità, Rome, Italy. ¹⁵Institute of Parasitology, Biology Centre, Czech Academy of Sciences, and Faculty of Sciences, University of South Bohemia, České Budějovice (Budweis), České Budějovice, Czech Republic. ¹⁶Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA. ¹⁷Department of Biology, The City College of New York, New York, NY 10031, USA. ¹⁸College of Marine Science, University of South Florida, Saint Petersburg, FL, USA. ¹⁹Biology and Health Sciences Department, Hendrix College, Conway, AR, USA. ²⁰Institute for Genome Sciences, Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD 21201, USA. ²¹Northern Gulf Institute, Mississippi State University, Mississippi State, MS 39762, USA. ²²Ocean Chemistry and Ecosystems Division, Atlantic Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric Administration, Miami, FL 33149, USA. ²³Data Collaboration Center, Critical Path Institute, Tucson, AZ, USA. ²⁴ANSES, Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail - Laboratoire de Ploufragan-Plouzané, Unité Génétique Virale de Biosécurité, Ploufragan, France. ²⁵Anses, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR VIROLOGIE, Laboratoire de Santé Animale, 94700 Maisons-Alfort, France. ✉email: fatima.esperancajorge@otago.ac.nz; nolwenn.dheilly@anses.fr</footnote>
microbiota. We also propose adding the term 'relationship to other packages' to all environmental packages across the domains of life, to allow for integrated analysis of symbiont and host microbiota by linking metadata elements across environmental packages. This will allow users to nest a package within another package, and to identify replicates. This added feature is pivotal for the study of the microbiome of symbionts that are themselves nested within a host, reflects the inherent nestedness of all ecosystems and will facilitate reports of complex sampling design from a myriad of environments.

Collecting relevant metadata (data describing data) is now widely recognized as critical to contextualize samples and increase their reusability and reproducibility [9–11]. The Genomic Standards Consortium (GSC, https://gensc.org) has developed and maintains a suite of minimal information metadata standards for describing sequence metadata (checklists) for genome (MIGS), metagenome (MIMS), marker gene sequences (MIMARKS), simple amplified genome (MISAG), metagenome-assembled genome (MIMAG), virus genomes (MIUVIG) and environmental packages for describing habitat-specific contextual data of the sampling environment [9, 10, 12, 13], collectively referred to as the Minimum Information about any (x) Sequence (MlXS) standard (ref. [9], https://gensc.org/mixs/).

The MlXS standards are used broadly across the microbiome research communities. These standards have been integrated into large scale microbiome projects (e.g. Human Microbiome Project, https://www.hmpdacc.org/), Earth Microbiome Project (https://earthmicrobiome.org/), Microbiology of the Built Environment (MoBE, https://www.microbe.net), microbiome bioinformatics platforms (e.g., QIME, Qiita, mothur, JGI GOLD, MG-RAST, EBI, NCBI) and are now required upon manuscript submission. A primary advantage of the MlXS standards is the collation of large aggregates of associated metadata that can be harnessed to uncover, and eventually comprehend, patterns of microbial diversity and ecology.

The MlXS-SA package was initially drafted during the 1st Parasite Microbiome project workshop that involved the contribution of members of the GSC in addition to microbial ecologists, parasitologists, pathologists and marine biologists [14]. Participants rapidly identified the need to incorporate information on the nestedness of symbiont-associated systems, and the absence within the MlXS host-associated package of descriptors of complex life histories of mutualistic and parasitic symbionts. Until now, researchers have either omitted this information or added research-specific symbiont-associated annotations, limiting significantly the potential to compare, combine and/or reuse data from different systems and studies. Whereas the MlXS-SA package was initially designed for the study of parasite-microbes interaction, the scope of the package was expanded to include non-parasitic symbionts. This addition is a necessary expansion due to the context-dependent nature of symbiotic interactions and the ability of a given symbiont to interact differently with different organisms. Notably, the resulting MlXS-SA package reduces the need to develop additional highly similar packages for different types of symbionts.

Symbiotic associations are generally classified as mutualistic (mutually beneficial association), commensal (beneficial association to one of the partners, but not harmful to the other), or parasitic (detrimental association to one of the partners) [15]. In the context of the symbiont-associated package, the term symbiont applies to macro and microorganisms that can establish a physical interaction with at least one other organism at some stage of their life cycle regardless of the nature and dependence of the interaction. As such, this definition also covers symbiotic organisms that establish facultative and accidental associations (e.g., dead-end hosts), not requiring evolutionary processes to explain their association, but excludes free-living organisms that establish a symbiotic relationship with another free-living

organism (e.g., flowers and bees). The MlXS-SA package presented herein has gone through an open and iterative review process engaging the GSC community and experts studying symbiotic organisms across various symbiont and host taxa.

Here, we present the selected list of metadata descriptors for symbiont-associated microbiota studies, including a subset of mandatory (M) terms that underpin metadata compliance (Table 1; Supplementary Information SI-1 contains all MlXS-SA items). In order to allow comparative studies of the microbiota of, sometimes closely related, free-living and symbiotic organisms, the MlXS-SA includes terms already found in the MlXS host-associated package. Thus, in MlXS-SA, the term "host" (when used alone) refers to the host of the biological sample which is the symbiotic organism. New terms were created to characterise the "host of the symbiotic host". We provide symbiont-associated package specific "Expected values" and "Examples". Changes to the package (addition of terms, modification etc.) can be proposed by the community by creating a ticket on the MlXS GitHub page (https://github.com/GenomicsStandardsConsortium/mixs).

Given the diversity of symbiotic interactions and that the nature and dependence of such interactions can be context-dependent rather than a fixed trait, it was crucial to define terms and provide value syntax that were inclusive for diverse types of symbioses and also across the symbiont life histories and transmission processes. For example, the term "host dependence" (a mandatory item) and "type of symbiosis" (a conditional item) are discrete but complementary items. While "host dependence" aims to provide a general characterization of the known type of host dependence for the symbiotic organism (e.g., facultative), "type of symbiosis" was specifically designed to further characterize the type of biological interaction established between the symbiotic organism and its respective host at the moment the biological sample was taken (e.g., mutualistic). As a result, the MlXS-SA package features mandatory and conditionally mandatory, and optional features that enable flexibility according to the knowledge of the study system at the time of sampling. Two examples of MlXS-SA-compliant metadata are provided in Supplementary information (SI-2), and the respective study designs are presented in Fig. 1. The examples refer to 16S rRNA gene studies of (a) the bacterial communities of the parasite *Coitocaecum parvum*, a trematode, across four of its life stages: the sporocyst, the metacercaria and the adult, as well as the free-living cercaria [16], and (b) of the leaves and roots of the parasitic plant *Orobanche hederae* and its ivy host, *Hedera* spp. [17].

While identical terms are often used in several of the 17 environmental packages currently available (https://gensc.org/mixs/), here we introduce three additional new terms: one is shared by several relevant MlXS environmental packages, and the two others will feature within the core MlXS package. The new term "observed host symbionts" provides a more comprehensive descriptor for the subject organism associations with smaller symbionts and it has been added to the host-associated, human-associated, plant-associated, human-vaginal, human-skin, human-oral and human-gut packages. The term "biotic relationship" has been added to the core package as a conditional descriptor of the relationship between the subject organism and other larger host organism(s). Finally, it appears necessary to include in the MlXS core a new term that takes into account the nested feature of most associations found in nature, such as host-symbiont-microorganism, in which multiple packages are necessary to describe the samples of the study (e.g., water, sediment, host-associated, and symbiont-associated). The proposed term "relationship to other samples" indicates the direct relationship between two samples from the same Bioproject, that are described in different environmental package(s). This proposed feature, still under development, will allow for integrated analyses of the microbiota of symbiotic organisms and their direct environment, even in the context of co-infections (e.g.,
<table><caption>**Table 1.** MlXS symbiont-associated environmental package representative terms, along with requirement status, description and MIXS IDs.</caption><thead><tr><th>MlXS Package</th><th>Metadata category</th><th>Package item</th><th>Req</th><th>Definition</th><th>MIXS ID</th></tr></thead><tbody><tr><td rowspan="10">Symbiont-associated</td><td rowspan="10">Symbiont-specific descriptors</td><td>host dependence</td><td>M</td><td>Type of host dependence for the symbiotic host organism to its host.</td><td>0001315</td></tr><tr><td>type of symbiosis</td><td>C</td><td>Type of biological interaction established between the symbiotic host organism being sampled and its respective host.</td><td>0001307</td></tr><tr><td>symbiotic host organism life cycle type</td><td>M</td><td>Type of life cycle of the symbiotic host species (the thing being sampled). Simple life cycles occur within a single host, complex ones within multiple different hosts over the course of their normal life cycle.</td><td>0001300</td></tr><tr><td>host life stage</td><td>M</td><td>Description of life stage of host.</td><td>0000251</td></tr><tr><td>mode of transmission</td><td>C</td><td>The process through which the symbiotic host organism entered the host from which it was sampled.</td><td>0001312</td></tr><tr><td>route of transmission</td><td>O</td><td>Description of path taken by the symbiotic host organism being sampled in order to establish a symbiotic relationship with the host (with which it was observed at the time of sampling) via a mode of transmission (specified in mode_transmission).</td><td>0001316</td></tr><tr><td>host number individual</td><td>O</td><td>Number of symbiotic host individuals pooled at the time of collection.</td><td>0001305</td></tr><tr><td>observed host symbionts</td><td>O</td><td>The taxonomic name of the organism(s) found living in mutualistic, commensalistic, or parasitic symbiosis with the specific host. For cases when the specific host of the sample is a symbiont this field should refer to other organisms it is associated with: e.g.: hyperparasite species X (parasite of the parasite).</td><td>0001309</td></tr><tr><td>host specificity</td><td>C</td><td>Level of specificity of symbiont-host interaction: e.g. generalist (symbiont able to establish associations with distantly related hosts) or species-specific.</td><td>0001308</td></tr><tr><td>host of the symbiont role</td><td>C</td><td>Role of the host in the life cycle of the symbiotic organism.</td><td>0001303</td></tr><tr><td>host cellular location</td><td>C</td><td>The localization of the symbiotic host organism within the host from which it was sampled: e.g., intracellular if the symbiotic host organism is localized within the cells or extracellular if the symbiotic host organism is localized outside of cells.</td><td>0001313</td></tr><tr><td>duration of association with the host</td><td>O</td><td>Time spent in host of the symbiotic organism at the time of sampling; relevant scale depends on symbiotic organism and study.</td><td>0001299</td></tr><tr><td>observed coinfecting organisms in host of host</td><td>O</td><td>The taxonomic name of any coinfecting organism observed in a symbiotic relationship with the host of the sampled host organism. e.g. where a sample collected from a host trematode species (A) which was collected from a host of host fish (B) that was also infected with a nematode (C), the value here would be (C) the nematode (species name) or (common name). Multiple coinfecting species may be added in a comma-separated list. For listing symbiotic organisms associated with the host (A) use the term Observed host symbiont.</td><td>0001310</td></tr><tr><td rowspan="3">Host of the symbiont descriptors</td><td>host of the symbiotic host common name</td><td>O</td><td>Common name of the host of the symbiotic host organism.</td><td>0001324</td></tr><tr><td>host of the symbiotic host local environmental context</td><td>O</td><td>For a symbiotic host organism the local anatomical environment within its host may have causal influences. Report the anatomical entity(s) which are in the direct environment of the symbiotic host organism being sampled and which you believe have significant causal influences on your sample or specimen. For example, if the symbiotic host organism being sampled is an intestinal worm, its local environmental context will be the term for intestine from UBERON (http://uberon.github.io/).</td><td>0001325</td></tr><tr><td>host of the symbiotic host environmental medium</td><td>O</td><td>Report the environmental material(s) immediately surrounding the symbiotic host organism at the time of sampling. This usually will be a tissue or substance type from the host, but may be another material if the symbiont is</td><td>0001326</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>MixS Package</th><th>Metadata category</th><th>Package item</th><th>Req</th><th>MIXS ID</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td>Definition external to the host. We recommend using classes from the UBERON ontology, but subclasses of 'environmental material' (https://purl.obolibrary.org/obo/ENV0_00010483) may also be used. EnvO documentation about how to use the field: https://github.com/EnvironmentOntology/envo/wiki/Using-ENV0-with-MixS. Terms from other OBO ontologies are permissible as long as they reference mass/volume nouns (e.g., air, water, blood) and not discrete, countable entities (e.g., intestines, heart).</td></tr><tr><td></td><td></td><td>host of the symbiotic host taxon id</td><td>O</td><td>NCBI taxon id of the host of the symbiotic host organism.</td></tr><tr><td></td><td></td><td>host of the symbiotic host subject id</td><td>O</td><td>A unique identifier by which each host of the symbiotic host organism subject can be referred to, de-identified, e.g. #H14.</td></tr><tr><td></td><td></td><td>Relationship to other samples<sup>a</sup></td><td>C</td><td>indicates the direct relationship with another sample from the same Bioproject. Accepted terms are: 'technical replicate of', 'after', 'before', 'next to', 'within', and 'contains'. Can be repeated to reveal relationship to many samples.</td></tr><tr><td></td><td></td><td>Biotic relationship</td><td>C</td><td>Description of relationship(s) between the subject organism and other organism(s) it is associated with. E.g., parasite on species X; mutualist with species Y. The target organism is the subject of the relationship, and the other organism(s) is the object.</td></tr><tr><td></td><td></td><td>Observed host symbionts</td><td>O</td><td>The taxonomic name of the organism(s) found living in mutualistic, commensalistic, or parasitic symbiosis with the specific host.</td></tr><tr><td></td><td></td><td>Other<sup>b</sup></td><td></td><td>0001309</td></tr></tbody></table>

Req requirements, O optional, C conditional, M mandatory, TBD to be determined.

aTerm may be slightly amended when included to all packages.

bTerm added to host-associated, human-associated, plant-associated, human-vaginal, human-skin, human-oral, and human-gut packages.

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Examples of study design for the sampling of microbes of symbiotic organisms, their hosts and environment. **a** Trematode *Coitocaeum parvum* different life stages (S1, S2, S3, S4) are reported with the MlxS-SA package. The microbiome of infected snail (H1), amphipod (H2) and fish (H3) hosts are reported with the MlxS host-associated package. In addition, the microbiome of environmental sediment (E1) and water (E2) from which these organisms were collected can be reported with MlxS-sediment and MlxS-water, respectively. The following relationships are reported: S1 "within" H1, S2 "within" E2, S3 "within" H2, S4 "within" H3, H1 "next to" E1, H1 "within" E2, H2 "within" E2, H3 "within" E2, E1 "next to" E2. **b** Angiosperm *Orobanche hederae* (S5, S6) parasitizing a host plant (P1, P2, P3) is reported using the included MlxS-SA and MlxS-PA (plant-associated) packages respectively. In addition, the MlxS-soil package is used to report corresponding soil samples. The following relationships are reported: S6 'within' P1, S5 'next to' S6, P1 'next to' P2, P1 'next to' P3, P2 'next to' P3, P1 'within' E3, P3 'within' E4, E3 'next to' E4.</figcaption></figure>

symbiont-associated SA1234 is "within" host-associated sample HA8974, "next to" symbiont-associated sample SA7890). This feature will also benefit other studies by providing ecologically-relevant contextual information (e.g., host-associated HA2567 is "within" environmental water sample W1234, "next to" host-associated sample HA5679, 'next' to environmental soil sample S5897). In conclusion, it is our hope that the MlxS-SA, together with the new terms, will enable researchers to better conduct integrated analyses of multi-level biological systems with the ultimate goal of better understanding the role of microbes associated with symbionts.

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## AUTHOR CONTRIBUTIONS

NMD conceptualized the MlxS package. All authors contributed to the writing and design of the MlxS-SA package. FJ and NMD prepared the manuscript. All authors agreed with the final version of the package and manuscript.

## COMPETING INTERESTS

The authors declare no competing interests.

## ADDITIONAL INFORMATION

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s43705-022-00092-w.

**Correspondence** and requests for materials should be addressed to Fátima Jorge or Nolwenn M. Dheilly.

**Reprints and permission information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## ACKNOWLEDGEMENTS

This contribution is part of the Parasite Microbiome Project. The authors sincerely thank the Gordon and Betty Moore Foundation for fully sponsoring the 1st Parasite Microbiome project workshop, and for supporting Open Access publishing. FJ was funded by a grant from the Marsden Fund (Royal Society of New Zealand) to RP (Principal Investigator) and NMD (Associate Investigator). The National Institutes of Health grants R01CA164719 (PJB), and R01AI144016-01(LJK). VH was supported by the FEDER grant InfoBioS n°EX011185 (région Val de Loire). JMM acknowledges support from award OCE-1933285, National Science Foundation, USA. ML is part of the PARADISE consortium, supported by funding from the European Union's Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme. JL was supported by the ERD funds (16_019/0000759).

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