www.dnaqua.net

ContactProf. Dr. Florian LeeseUniversity of Duisburg-EssenAquatic Ecosystem ResearchUniversitaetsstr. 5, 45141 Essen, Germanyemail: chair@dnaqua.netphone: +49.201.183.4053

EU COST Action CA15219

April 2018

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In this issue:

· Editorial

· Announcements

· Working groups

· General reports

· STSMs

· Representation ofDNAqua-Net

· Publications

http://www.dnaqua.net

Editorial

Dear DNAqua-Net members, dear reader,

I am not completely sure when I gave up my ambitions to read every new study on DNA / eDNA metabarcoding. It must have been already over a year back. The tech-nical revolution in the field boosts scientific output, delivering floods of new findings from research groups all around the globe. The challenge now is to not become frustrated, but to see the potential in the rapid developments and team-up with partners in order to connect the many new findings and derive overarching principles.

To a major extend, this is what DNAqua-Net is for; and while undoubtedly the organisation of the network takes away even more time from my schedule that I could potentially use to read new studies, I am quite happy to not do so and instead invest my resources to connect the different actors in the field and retrieve the knowledge in an often much more condensed and filtered form through the network. The dialogue between the many experts in the network from Europe and beyond allows us to find consensus on

many aspects that can be ‘dis-tilled’ into good practice guidance documents. Sometimes consensus is very close and graspable. Other times it is still a long way off and more coordinated research needs to take place to do this.

And this is going on - in just the last 12 months, we have initiated about 20 Short Term Scientific Missions (STSMs) from our network, have hosted several workshops in different European countries and will continue to do so in the future.

But while most of this takes place largely on academic ‘play-grounds’, we are bridging the science-application gap more and more in order to improve biodiver-sity assessments. Although meth-ods continue to evolve, there are already excellent routines identified by members that can serve as guid-ance for practical implementation.

This has been shown by the report of Working Group 5 (“Im-plementation strategy”) with a specific focus on the European Water Framework Directive (2000/60/EC). The results have just been published in the journal “Water Research”; and while the report stresses the need for further refinement and parallel compari-sons of traditional and DNA-based assessments, it already shows that for Biological Quality Elements, such as fish, eDNA techniques are already quite advanced and ready for complementing traditional assessments.

A recurring theme that has emerged in all the different Work-

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Announcementsing Group meetings as well as in most of stakeholder meetings we have been invited to is “standard-isation”. We realise that especially in the quickly emerging field of DNA tools, it is of pivotal impor-tance to agree to standards in order to allow comparability of the data.

We are excited that the Europe-an Committee for Standardisation (CEN) has also identified this as a topic of great importance and members of our network together with CEN have come up with a proposal for the establishment of a new permanent working group on “DNA methods” as part of the Technical Committee TC230 (“Water Analysis”). We will keep you informed about the outcome (see article in the newsletter).

Now, together with the ‘Core-Group’ members, we are eagerly putting together last documents for the Work and Budget Plan for the upcoming Grant Period #3 starting in May 2018. Under the umbrella terms “Collect and Connect” Grant Period 3 will specifically invest in mobilising researchers and ideas, connecting countries, different disciplines, and work toward further strategies to improve biodiversity assessments with DNA-based tools.

For now, I hope you enjoy read-ing about the progress of DNAq-ua-Net in this newsletter and I am looking forward to meeting many of you in the upcoming meetings in Hungary, Pécs in June 2018.

Prof. Dr. Florian Leese(Chair of DNAqua-Net)

General

- Unfortunately, Smolenice Castleis already fully booked in June2019. If any of you would like tohost and organise our fifth Man-agement Committee Meeting(MC5) in June 2019, please sendan E-Mail tomanager@dnaqua.net

- Remember the possibility forEarly Career Researchers fromInclusiveness Target Countries toactively join a Conference.Please apply for an ITC Confer-ence Grant via E-Mail atchair@dnaqua.netFurther information: http://www.cost.eu/media/cost_stories/conference-grants-new-vade-mecum-2017

- The Nagoya protocol and theconsequences for DNA-basedbioassessment across countrieshas many legal issues to consider.Especially during MC3 meetingin Sarajevo, this was a topic. Arecent book published throughPensoft deals with this issue, ex-plicitly answering questions thatour community of DNA barcod-ers need to know:https://ab.pensoft.net/articles.php?id=22579

- We made some changes on ourHomepage. Have a look:http://dnaqua.net/

Events

- Our next Management Com-mittee Meeting (MC4) will takeplace on June 12th, 2018 inPécs, Hungary. Following somegeneral discussion about theprocedure of a MC Meeting wehad in Sarajevo, we decided tochange it a bit. This time we willhave the administrative things(like reports, votes) done in themorning and will already startwith scientific work in form ofoverview talks in the afternoon.On June 13th and 14th 2018every Working Group will havea Meeting in Pécs. For moreinformation about these meet-ings please contact your WorkingGroup leaders.

- Thanks to Patricia Mergen, a ses-sion was accepted at the TDWG/SPNCH Conference in Dune-din, New Zealand from August25th to September 1st, 2018. Itwill be about “Collections andData in an Uncertain World”.

- From June 26th to 30th the 25thInternational Diatom Sympo-sium will take place in Berlin,Germany. Working Group 2 willhave a diatom workshop therefrom June 27th to 29th. Thanksto Jonas Zimmermann it is alsopossible to have some peoplegoing there with an ITC Confer-ence Grant.

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Endre Willassen on taxonomic-feedback-loops

Working group reports

Braga working

Working Group 1: DNA Barcode References

Thanks to Filipe Costa and his marvellous organising team, we had very nice meeting and produc-tive workshop from 24th to 25th January 2018 in the brand-new building of the Institute of Science and Innovation for Bio-Sustain-ability at University of Minho in Braga, Portugal.

We were welcomed by Fernan-da Cassio, head of the Centre of Molecular and Environmental Biology, who presented us with a number of interesting and relevant projects at the University of Min-ho. TorbjørnEkrem (NTNU Uni-versity Museum, Norway) opened the meeting with a reminder of the goals of WG1. Professor Endre Willassen (University Museum of Bergen, Norway) followed with a talk on observations made during the development of the barcode reference library on marine in-vertebrates in Norway. A central message was that continuous curation through the taxonom-ic-feedback-loop is necessary to for the quality and usefulness of a reference library. The following discussion on quality aspects of barcode reference libraries was led by Alexander Weigand (University of Duisburg-Essen, Germany) and resulted in some important points

that can certainly be repeated in the planned paper. Before the actual work started, Fedor Čiamp-or (Slovak Academy of Sciences, Slovakia) gave an overview of the activity in WG1 and the nations that are represented in the current work.

The WG1 workshop was intended to provide consistent taxonomic checklists to perform gap-analyses of the barcode reference library for the European aquatic biota. Thirty-six experts attended the meeting, processing checklists from more than 19 countries of various (freshwater and marine) animal and plant groups used in the implementation of the Europe-an directives (WFD, MSFD).

The main workshop outputs:1. More than 30 taxon checklists

were produced.2. National checklists of 22 fresh-

water invertebrates were trans-formed into BOLD-formatted checklists (Hirudinea, Hyme-noptera, Lepidoptera, Megalop-tera, Neuroptera, Plecoptera,

Platyhelmithes, Annelida, Coleoptera, Heteroptera, Arachnida, Crustacea, Diptera, Mollusca, Odonata, Ephemerop-tera, Trichoptera, Pisces, Dia-toms, Vascular plants) including almost 8000 records.

3. National checklists of 4 marine groups were transformed into BOLD-formatted checklists (An-nelida, Arthropoda, Nemertea, Echinodermata) including more than 9500 records.

4. The generation of lists of syno-nyms was initiated.

5. Preliminary gap-analyses showed that in average around 50% species lack DNA barcodes, only 4 groups out of 24 showed more than 80% species coverage (these however represent less than 2% of the listed species/taxa).

The important deadlines:Since there are still national

checklists missing from several countries and we need to clarify the list from Spain, we decided to postpone final gap analyses by two weeks (Feb 12).

We also initiated joined work on the review article with prelim-inary title “DNA barcode refer-ence libraries for aquatic biota in Europe: Gap-analysis and recom-mendations for future work”. The draft was placed on Google Docs. We have agreed that the final draft will be ready by MC4 meeting in June 2018 (Pécs, Hungary).

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WG3 working in Innsbruck

Working Group 2: Biotic indices and Metrics

One main activity of WG2 during the last activity period was the joint writing of the WG2 review article “The future of biotic indices in the ecogenomic era: integrating DNA metabarcoding in biological assessment of aquatic ecosystems”.

The article is now under re-view. Several of our members have also been busy as co-authors of the Umbrella-article (Leese et al. 2018) and the WG5 review article (Hering et al. 2018). WG2 mem-bers were involved in eight STSMs (Anders Lanzén, Bonnie Bailet, Jasmina Kamberovic, Tristan Cordier, Jan Macher, Andreia Mortágua, Andrey Rozenberg).

Other ongoing projects involv-ing WG2 members are compar-ing traditional and eDNA based monitoring of invertebrates in the Nordic countries (led by University of Duisburg-Essen and SYKE) and in Belgium (cooperation of Univer-sity of Duisburg-Essen, Provincial Centre of Environmental Research and HoGent in Flanders).

Finally, WG2 is planning for several future activities, among them the designated workshops in Pécs (13-14 June 2018, fish, in-vertebrates), Berlin at IDS (26-30 June 2018, diatoms), and Dublin or Prague (late 2018/2019, micro-organisms & meiofauna).

Working Group 3: Field and lab protocols

Working Group 3 held a work-shop in Innsbruck, Austria (26-27

March 2018). The theme of the workshop, attended by 35 dele-gates, was the development and validation of assays for single-spe-cies detection using PCR-based approaches. The extent of labora-tory and field validation applied to published tests is highly variable, and this makes it difficult for po-tential end users to know whether any given assay is suitable for use. We heard talks from delegates describing various different assays and methodological comparisons, and then considered the key steps in a ‘gold standard’ assay valida-tion, ranging from in silico testing to statistical determination of detection rates in natural settings.

On day 2, we split into three task groups to begin work on the three main workshop outputs:- a set of guidelines for assay devel-

opment and validation, building on Goldberg et al., 2016. This was drafted during the workshop and will be refined by a task group going forward.

- a list of agreed definitions of key terms relating to PCR-based species detection. The list of terms was compiled during the meeting, and definitions will be debated henceforth!

- a validation scale to commu-nicate to end users the level of validation applied to a particular assay. For each validation level, this tool will outline the remain-ing uncertainties that should be taken into account by the user, and describe how results should be interpreted if the assay is used. During the workshop, we decid-ed on the 5 levels on the scale and the broad validation steps that will have been undertaken to achieve each level. These will be refined by a task group going forward.

We were also grateful to Roger Sweeting for joining us to explain the ins and outs of CEN stand-ardisation so that we can consider how formal standardisation might be applied to PCR-based detec-tion of key species. A huge thank you to Michael Traugott, Caro-line Pichler and the University of Innsbruck team for all their hard work in arranging and hosting this meeting.

Several DNAqua-net members attended the UK DNA Working Group meeting in Salford, UK (5-6 December 2017). This work-ing group consists of UK-based end-users and policy-makers as well as academics and practition-ers of DNA monitoring. Bernd Hanfling and Micaela Hellstrom presented the results from their STSM work on fish eDNA me-tabarcoding, Kat Bruce presented an overview of CEN standardi-sation process, and several other WG3 members presented their research.

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Innsbruck participants

Berlin workshop participants

It was great to see DNAqua-net so well represented in this forum and also to see the appetite among UK policy-makers to adopt DNA methods and to ensure that they are intelligent users of new tools. Since the UK is so active in this regard, we feel it is important to facilitate communication in both directions between DNAqua-net and the UK DNA working group and are encouraging the UK Environment Agency to become more involved with DNAqua-net activities.

In other WG3 news, work continues on experiments to de-termine the viability of extracting DNA from preservation buffer used to store samples of benthic macroinvertebrates. Jan Macher and Rosie Blackman hope to pres-ent preliminary results soon. This was one of the key work items that emerged from the WG3 workshop on macroinvertebrates in Sarajevo.

Working Group 4: Data Analysis and Storage

Working Group 4 deals with two main lines of work in this period: unsupervised OTU (Operational Taxonomic Unit) tables and HPC (High Performance Computing).

The first topic is a review and test of pipelines to obtain OTU

tables using unsupervised algo-rithms, without any taxonomic ref-erence library identified to species level. The tests will be performed on different datasets (freshwa-ter fish, freshwater diatoms, and marine meiofauna) on different markers with different lengths, and using the most commonly applied pipelines. Part of the tests are performed in collaboration with a STSM (Short Term Scientific Mis-sion). The tests will be performed to compare speed, reliability, and accuracy of the procedures for metabarcoding.

The second topic is a review of existing accesses to HPC, with the aim of implementing full-scale tests on the same datasets used in the first topic. One aim is to get clusters using algorithms without any heuristic search (shortcuts to speed the process but that may produce bias), in order to validate the heuristics in the pipelines (or not ...) for OTU building.

As a first step, we are connect-ing the tools we use in DNAq-ua-net with EGI (European Grid Infrastructure), aiming at access to intensive computing when needed.

Working Group 5: Implementation strategy and legal issues

WG5 has prepared a paper enti-tled “Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive” (authored by Dan-iel Hering, Angel Borja, J Iwan

Jones, Didier Pont, Pieter Boets, Agnès Bouchez, Kat Bruce, Stina Drakare, Bernd Hänfling, Maria Kahlert, Florian Leese, Kristian Meissner, Patricia Mergen, Yorick Reyjol, Pedro Segurado, Alfried Vogler, Martyn Kelly). It will appear in due course in Water Research (see: chapter “Publica-tions”).

Other activities of WG5 include the preparation of an online survey on DNA-based assessment meth-ods and monitoring programmes. It was originally planned to already go online last year – however after several delays it will now be final-ised within the next week.

On February 8th, 2018, DN-Aqua-Net Working Group (WG) 5 organised a workshop at Berlin Botanical Garden and Botanical Museum (BGBM). Local hosts were Jonas Zimmermann and Regine Jahn. The aim of this work-shop was to discuss what and how to standardise procedures of new genetic tools for routine aquatic bioassessments. An obvious risk is that without standardisation, data created with the new genetic tools will lack both backward compat-ibility and not be comparable to data created with future advances

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Active discussions on “what” and “how” to standardise

General reports

in technology. An obvious plat-form to link questions regarding of European-wide standards to is the European Committee for Stand-ardisation (CEN), https://www.cen.eu/. Already in 2017, CEN Working Group 2 on “Biological Methods” of the Technical Com-mittee 230 (CEN/TC230/WG2) had invited Prof. Florian Leese to start the discussion about the need for standards in this area and the Berlin workshop was the direct extension of this. Together with DNAqua-Net experts from Fin-land, Belgium, UK, Switzerland, Germany and France, as well as members of CEN (Roger Sweet-ing) and a representative of the German standardisation authority DIN (Andreas Paetz) we had a very constructive discussion on items, processes as well as Quali-ty Assessment / Quality Control that require standardisation. The participants also agreed that stand-ards should be developed in strong European cooperation to allow general consensus on standardised best practices without impeding on further technological development.

More discussions and work are needed, to identify and turn mature enough procedures in the pipeline of genetic tools into work-ing standards. To facilitate this process on a permanent basis, the DNAqua-Net participants with the help of CEN experts Roger Sweeting and the Chair pro tem of the potential new WG28, Kris-tian Meissner drafted a proposal for the implementation of a new permanent CEN Working Group (WG28) on “DNA methods”. We deeply appreciate the constructive dialogue with CEN and will con-tinue to provide input specifically through DNAqua-Net WG 3 (Lab and Field methods).

Ana Rotter @ training school for communication in COST

On 9.2.2018 I participated at a great course organised by COST Academy entitled “Working with the media. Mastering media interviews”. The course was led by Jacki Davis, an experienced EU journalist.

Communication managers from various COST Actions were first invited to briefly present their respective Actions and try to be as brief, informative and interesting as possible. We then proceeded to the formal part of training, where Jacki gave us important pieces of advice that will help us in prepar-ing ourselves for future conversa-tions with the media.,

I will list some important take-home messages:Remember these questions (in this

order): WHAT, WHY, WHO, WHEN, WHERE and HOW. This is the sequence in which you have to prepare any speech, outreach, contact, press release or similar.

The worst science communicators are people who don’t like/trust journalists.

You should always strive to make the content interesting. If you don’t spice it up, the journalists will. And don’t get mad at them, we tend to spice things up as well, especially when prepar-ing project proposals where we promise to save the world

Keep the story simple and try to avoid using jargon words. It makes unnecessary barriers between you and the target audi-ence and your chances of success (spur of interest by the target audience) will be lower.

TV: remember, people will remem-ber only 1-2 things you say. So you better prepare yourself and make an effort to highlight these 1-2 things

PRESS RELEASE: keep it short (1 page) otherwise journalists will abbreviate. Pictures matter! Include the background of the study. Again, avoid using jargon terminology or acronyms.

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SOCIAL MEDIA: you can repeat the same message on Twitter more than once, while on Face-book you can write a story only once.

BROADCAST: take bullet point-ed sheet of important messages with you as a cheat sheet. Try to speak in complete sentences, otherwise you will risk being taken out of context. If they keep asking the same question over and over again, it means they are probing you or you are not giving a clear answer and they do not understand.

Hostile questions: be polite and do not repeat the negative state-ments.

Secret weapon: confidence!

After the course and filled with new obtained knowledge, we repeated the introduction round of presentations of individual Actions. This time around, every Action was fascinating, interesting, state-of-the-art and we wished we could be a part of all of them!

News

(e)DNA-based biomonitoring of the Danube – Our cooper-ation with the ICPDR

The International Commission for the Protection of the Danube River (ICPDR) organises an inte-grative monitoring of the Danube River every 6 years. The next Joint Danube Survey (JDS4) will take

place in late summer 2019, collect-ing biological, microbiological, hy-drological as well as chemical data for different sampling sites along the main channel of the Danube and within several countries. For the first time, effect-based tools as well as (e)DNA-methods will be integrated in the monitoring. For the latter, the DNAqua-Net consortium will act as the central partner.

Since the first announcement in the last newsletter, the DN-Aqua-Net leaders for the specific JDS4 molecular biomonitoring subgroups now have been found:

JDS4DNA-Fish incl. JDS4D-NA-eDNA (Didier Pont & Emre Keskin)

JDS4DNA-MZB (Florian Leese)JDS4DNA-Diatom

(Jonas Zimmermann)JDS4DNA-Barcode (Jonas Astrin)

If you are interested in joining any of the groups or would like to have more information, please refer to https://tinyurl.com/JDS4D-NA or email to Alexander Weigand (alexander.weigand@uni-due.de). Some of you have already stated your interest to participate, and you should be listed as team mem-bers in one of the aforementioned subgroups in the document. There will be also a close cooperation between a Microbiology-team from Austria and our eDNA sam-pling-team.

To allow preliminary training for some members of the JDS4 na-tional sampling teams, a session on “(e)DNA-conformable sampling” will be organised during a larger WG3-organised workshop event in Bucharest (Romania; Local Or-ganisation by Marieta Costache) during the last week of October 2018. Also, the Management Committee (during MC3 in Sara-jevo) agreed to support the JDS4 national sampling teams in 2019 with Short-term Scientific Missions (STSMs), i.e. participants from DNAqua-Net joining the regional samplings for the Danubian biota to assist in molecular handling.

STSMs

Overview

The third call for Short-Term Scientific Missions (STSMs) in Grant period (GP) 2 was the most dynamic so far. Out of 11 applica-tions that were submitted, STSM committee and leaders of working groups positively graded ten that were ultimately supported. More intense involvement of ITCs was also noticed, both as home (50% of all successful applicants) and host (40%) countries. Most of the STSMs targeted objectives of Action working groups 1 and 2. Topics of supported applications were diverse: from establishing platforms and gathering DNA metabarcoding data for diatoms and macroinvertebrates to develop new eco-indices in ITCs (Portugal, Bosnia and Herzegovina, and

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Malta) over Development and application of supervised ma-chine learning for bioassesment of sediments impacted by differ-ent sources of contamination; Monitoring offshore oil-drilling platforms for ecological quality status assessment; Development of a bioinformatic pipeline to assess stressor responses in fungal com-munities from metatranscriptome data; to Development and applica-tion of supervised machine learn-ing for bioassesment of sediments impacted by different sources of contamination.

The first STSM call in GP3 for missions occurring between 19th of May 2018 - 1st of March 2019 is now open.

Detailed information on how to apply can be found online at http://dnaqua.net/stsms/.

Important dates for the upcoming evaluation round are:

Deadline for applications to be submitted for next evaluation: May 11th, 2018.

Notification of application outcome: May 18th, 2018.

Period of STSM: between 19.05.2018. and 01.03.2019.

Reports

Isabel Fernandes

“Standardisa-tion of sampling methods for assessing fun-gal diversity in freshwaters with metabarcoding”

The STSM was hosted by Profes-sor Guy Woodward at Imperial College London, United Kingdom. The host institution is currently running a pond mesocosm exper-iment (http://www.imperial.ac.uk/silwood-park/research/silwood-lte/mesocosm/), and one of the goals is to standardise measures of mi-crobial community structure.

The main goal of this STSM was to standardise methodologies for assessing fungal diversity in freshwaters with metabarcoding approaches. To achieve that, two tasks were proposed: 1. Isolation of aquatic fungi from

different substrates from the pond mesocosms and generation of ITS barcodes; This task aligns with the objectives of the WG1: DNA Barcode Reference Data-bases.

2. Execution of an experiment to evaluate which sampling substrates allow the recovery of highest fungal diversity using metabarcoding approaches; This task aligns with the objectives of the WG3: Field and Lab Pro-tocols, which aim is to discuss/evaluate current protocols for

sampling, processing, and labora-tory approaches to assess diversi-ty with eDNA/metabarcoding.

Jasmina Kamberovic

“Preparation of the pilot project proposal on metabarcoding and morphologi-cal identification to assess diatom

diversity in poorly explored Dinar-ic rivers”

The main purpose of this STSM was to achieve a cooper-ation between researchers from Bosnia and Herzegovina (Inclusive Target Country) and researchers from Sweden having long experi-ence in implementing the Water Framework Directive (WFD), with the aim of an exchange of expertise and of preparing a mutual pilot project proposal.

The specific aims of the STSM were: (i) a knowledge exchange on eDNA metabarcoding method-ology on diatoms, (ii) the devel-opment of a mutual pilot project proposal on metabarcoding and morphological identification to assess diatom diversity in poorly explored Dinaric freshwater rivers, and (iii) the evaluation of the di-atom reference community in the Bosnian freshwater ecosystems as a contribution to the development of the index.

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Andreia Montagua

“DNA metabar-coding approach as a complemen-tary technique for assessment of Portuguese rivers – contri-

bution to an Eco-genetic index”The main purpose of this STSM was the training on DNA metabar-coding for diatomcommunities in a WFD-biomon-itoring aim. This included 1) finali-sation of the protocolsfor preparation of HTS libraries (DNA extraction and PCR ampli-fication) for DNAsequencing in a sequencing plat-form (Illumina MiSeq), 2) analys-ing the sequence dataand 3) computing bioindicator metrics. The main aim is to apply this approach in parallelwith the classical morphological approach to diatom samples from Portuguese rivers.This work should develop into a new biometric index, which aims to complement thetaxonomic identification of dia-toms (using the light microscope) for rivers’ assessmentpointed out in the Water Frame-work Directive (WFD).

Tristan Cordier

“Monitoring offshore oil-drilling platforms: prospecting existing biotic indices for ecological quality status assessment”

The purpose of the STSM was to strengthen and foster new collabo-rations between the AZTI research centre, in the Spanish Basque Country, and the Jan Pawlowski group at the University of Gene-va, Switzerland. The AZTI holds long-standing and internationally recognised expertise for the eco-logical quality status assessment in marine environments. The institute pioneered research in the field of eDNA metabarcoding data applied for ecological quality assessment, with the work of Eva Aylagas (PhD student supervised by

Dr. Naiara Rodriguez-Ezpeleta and Dr. Angel Borja) that led to the development of the gAMBI index for macro-invertebrates (Aylagas et al., 2014) and mir-croAMBI for bacteria (Aylagas et al., 2017). Dr. Anders Lanzén, an experienced microbial ecologist and bioinformatician, joined the AZTI institute in January 2018. He holds strong expertise in the analysis of meta-omics data and was recently involved in projects aiming to better assess the impacts of offshore oil-drilling platforms in the North Sea (Lanzén et al., 2016). Our group was among the first ones to apply eDNA metabar-coding approach for the monitor-ing of Salmon farms in Norway (Pawlowski et al., 2014; 2016; Cordier et al., 2017). We recently got involved in a biomonitoring project for oil-drilling platform in the Adriatic Sea.

The main objective of the STSM was to analyse the dataset recently collected for the latter

project on three oil-drilling plat-forms.

More specifically, the objectives were to:1. Use multivariate statistics to

explore both alpha and beta-di-versity of OTUs (taxonomically assigned or not), to test whether there is a change of communities around the three platforms.

2. Study the explanatory potential of physico-chemical parameters on the changes of communities.

3. Measure the amount of meta-zoan assigned OTUs from the Adriatic that are present in the gAMBI database, and the bac-terial assigned OTUs present in the microAMBI.

4. Compute the gAMBI from the metazoan OTUs and microAM-BI from bacterial OTUs, to test whether theses indices are reflecting the physico-chemical parameters well.

A side and exploratory aim of the STSM was to evaluate the possi-bility to analyse various datasets collected by both of the groups together in a “meta-analysis” to test the applicability of machine learning techniques across different geographic regions.

Anders Lanzén

“Development and application of supervised machine learning for bioassesment of sediments impact-ed by different sources of contami-nation (#39578)”

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The purpose of the STSM was to strengthen the scientific and stra-tegic collaboration between AZTI (Basque Autonomous Community, Spain) and the research group of Prof. Jan Pawlowski at the Uni-versity of Geneva. The Pawlowski group were among the first to ap-ply metabarcoding of environmen-tal DNA (eDNA) for ecological monitoring and evaluate its use as a routine diagnostic complement-ing quality indices based on visual inspection of macrofaunal diversi-ty. Their work has focused mainly on monitoring of aquaculture sites (Pawlowski et al. 2014) but recent-ly also on offshore oil extraction.

Dr. Tristan Cordier in the same group has successfully explored the use of machine learning (ML) for accurate quality classification of metabarcoding data (Cordier et al. 2017). This approach can bypass taxonomic classification or implicit taxon distribution models and has great potential for rou-tine use in large scale monitoring programmes.

Thus, it is highly relevant for ongoing research activities at AZTI where I participate, in-cluding routine monitoring of the impact of residual and industrial contamination on benthic ecosys-tems, and the development of new biotic quality indices, based on metabarcoding of benthic meio- and microfauna. AZTI have long standing experience with tradition-al and metabarcoding-based assess-ment of ecological quality (see e.g. Borja et al. (2000); Aylagas et al. (2014); Aylagas et al. (2017)) and

will certainly be able to contribute to the further development of these techniques.

The specific objective of this STSM was to learn, adapt and evaluate the analysis workflow developed by Cordier et al. for two existing metabarcoding datasets, using the small subunit ribosomal RNA (SSU rRNA) as taxonomic marker. The first dataset, (BMN – Basque Monitoring Network) is from estuarine and coastal benthic communities along the Bay of Bis-cay, at sites established as part of a long-term monitoring programme (Borja et al. 2016). The second (OE – Oil Extraction) is from Norwegian offshore oil drilling sites analysed in a recent proof-of-concept study (Lanzén et al. 2016) that will be continued in collabo-ration with Uni Research and the University of Bergen (UiB / UR), Norway. A secondary objective of the STSM was to discuss metabar-coding analysis applied to offshore oil drilling sites, exchange experi-ences related to this and establish a collaboration including both AZTI, UiB /UR and UNIGE.

Another objective added during data preparation to the STSM was to analyse datasets collected in sep-arate projects by the two groups, in a “meta-analysis”. This evaluates the applicability of ML techniques across distinct geographical re-gions.

Neus Marí-Mena

“Testing differ-ent pipelines to obtain OTU tables from mas-sive sequencing”The main purpose of

this Short Term Scientific Mis-sion (STSM) was to carry out an examination of different bioinfor-matics pipelines to analyse DNA metabarcoding datasets generated by high-throughput sequencing. In order to hold the potential to assess biodiversity of freshwater ecosys-tems more reliably in DNA me-tabarcoding studies, standardised pipelines are crucial. This is one of the goals of the working group 4 (WG4) of the DNAqua-Net.Given the wide range of options for data processing and parameter settings existing for the sequences clustering step of the DNA me-tabarcoding bioinformatics work-flow, we decided to evaluate the suitability of the different methods by describing the effects of dif-ferent algorithms and parameter choice on the results, i.e. on the assignment of sequences to OTUs across samples.The dataset used to test the differ-ent pipelines was generated prior to the STSM at the company All-Genetics & Biology S.L. The data consisted of 18S reads generated from marine meiofauna samples with the Illumina MiSeq PE300 technology (PE300).

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Representation of DNAquaNett

Tomasz Rewicz

“Towards filling the gap in Euro-pean Freshwater Macroinver-tebrate DNA barcoding – a comprehensive

project in white spot, Malta”I selected the Malta College for

Arts, Sciences and Technology in Malta, to host this STSM, since it is one of the ‘white spots’ in European map of checklists and barcode libraries especially for rare, endemic and so far unknown spe-cies, will be very useful for WG1. It could also be a great chance for the transfer of knowledge and ex-perience from me to the local team in Malta and an opportunity to build an international collaborative group for this project, as a pilot project and a core group, which may induce further spinoff pro-jects based on this year findings. The specific goals of the current STSM were: a) taking comparative quantitative bulk samples (AQEM, assessment types) from many in-termittent streams at the same site and at the same time in parallel for morpho-taxonomical and metabar-coding identification; b) access to comprehensive invertebrate sam-ples from all available freshwater water bodies in that country; c) learn how to collect AQEM based (assessment type) quantitative samples; d) learn how to collect water samples (filtering in the field) for further eDNA metabarcoding

studies; e) learn how to collect algae samples.

Andrey Rozenberg

“Development of a bioinformatic pipeline to assess stressor responses in fungal communities from meta-transcriptome data”

The aim of the proposed STSM was to contribute to the project “Mechanisms and thresholds of fungicide effects on microbial communities and organic matter processing” led by Dr. Romana Salis and supervised by Prof. Dr. Florian Leese (University of Duis-burg-Essen) and Prof. Dr. Ralf B. Schäfer (University Koblenz-Lan-dau). The project is aimed at answering the two questions: who (what groups/species/OTUs) contribute to leaf litter decomposi-tion and what they do (what genes are recruited in the process), with the main focus on hyphomycete fungi, the key members of the community. An elaborate experi-mental design puts these questions in the context of time (changes in the community as decomposition progresses) and anthropogenic stressors (control vs. pesticides). Besides morphological identifica-tion of the taxa, metatranscriptom-ics will be used to target both of the questions. The STSM proposed here aims at helping to design and develop a pipeline for the analysis of the metatranscriptomic data.

At this phase of the project development, the main focus has been on planning and initial

development of the metatranscrip-tomic workflow. The main topics discussed during the STSM have been:- big decision to be made: whether

metatranscriptomics methods can also be used for taxonomic identification

- big decision to be made: the need for custom reference transcrip-tomes

- revision of the available knowl-edge on hyphomycete species expected in the experiments

- inspection of the potential utility of the available barcoding and genome databases for hyphomy-cetes and related species

- choice of the barcoding fragment- strategy for functional gene char-

acterisation

Simon Vitecek @ EGU GA in Vienna

From April 8th until 13th, the General Assembly of the European Geosciences Union (EGU) is held in Vienna. In a session dedicated to ongoing COST Actions, the unique challenges and potential of DNAqua-Net were presented.

The EGU is one of the largest scientific meetings, and our mis-sion to develop a comprehensive pan-European molecular bio-

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Participants of the Global Omics Workshop in Bremen

Publicationsassessment were well received by a highly diverse audience. Engaging with complementary ecosystem sciences is one of the key strategies of DNAqua-Net, and our rep-resentation at this event will foster future collaborations.

Florian Leese @ Global OM-ICS Observatory Workshop

Florian Leese was invited to represent DNAqua-Net at a 3-day workshop hosted at the Max Planck Institute for Marine Mi-crobiology, Bremen (Germany). The workshop initialised a “Global Omics Observatory Network” (GLOMICON) to better coordi-nate multiregional, long-term, and omically enhanced observation activities.

Local host and DNAqua-Net member Pier Luigi Buttigieg (Al-fred Wegener Institute) – as well as Jörg Peplies (RiboCon GmbH), Felix Janssen (Alfred Wegener Institute), and James Macklin (Ag-riculture and Agri-Food Canada) – organised the meeting to bring together an international consorti-

um of prominent omics research-ers (affiliated with, e.g., GEO BON/MBON, TARA Oceans, NEON, NOAA, the GSC, the Earth Microbiome Project, and the Genomic Observatories Network). Participants discussed practical approaches to enhance the inter-operability and coordination of long-term OMICS observation activities.

Following a day of plenary presentations, breakout sessions addressed questions such as “How can a global OMICS observatory network cope with rapid techno-logical change?” and “How can we ensure that distributed metadata is consistent, harmonised, and exchangeable?”

The group’s collective answers to these and related questions will be published as part of a meeting report. It became obvious that there are many synergies between GLOMICON and DNAqua-Net activities; we will strive to organise a follow-up meeting in the near future in order to develop sustain-able mechanisms to continue such discussions even after the formal end of DNAqua-Net.

More information about the Bremen workshop is available here: https://www.atlantos-h2020.eu/events/workshop-on-enhancing-om-ics-observation/

Two main publications from the network were published within the last 3 months 1) Umbrella paper and 2) WG5 paper.

All other publications by partic-ipants of the network can also be found on the website.

Umbrella paper

“Why We Need Sustainable Net-works Bridging Countries, Disci-plines, Cultures and Generations for Aquatic Biomonitoring 2.0: A Perspective Derived From the DNAqua-Net COST Action”https://www.sciencedirect.com/sci-ence/article/pii/S0065250418300011

The Abstract of the Paper is:Aquatic biomonitoring has

become an essential task in Europe and many other regions as a con-sequence of strong anthropogenic pressures affecting the health of lakes, rivers, oceans and ground-water. A typical assessment of the environmental quality status, such as it is required by European but also North American and other legislation, relies on matching the composition of assemblages of organisms identified using mor-phological criteria present in aquat-ic ecosystems to those expected in the absence of anthropogenic pres-sures. Through decade-long and difficult intercalibration exercises among networks of regulators and scientists in European countries, a pragmatic biomonitoring approach was developed and adopted, which

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now produces invaluable informa-tion. Nonetheless, this approach is based on several hundred different protocols, making it susceptible to issues with comparability, scale and resolution. Furthermore, data acquisition is often slow due to a lack of taxonomic experts for many taxa and regions and time-con-suming morphological identifica-tion of organisms. High-through-put genetic screening methods such as (e)DNA metabarcoding have been proposed as a possible solution to these shortcomings. Such “next-generation biomonitor-ing”, also termed “biomonitoring 2.0”, has many advantages over the traditional approach in terms of

speed, comparability and costs. It also creates the potential to include new bioindicators and thereby further improves the assessment of aquatic ecosystem health. Howev-er, several major conceptual and technological challenges still hin-der its implementation into legal and regulatory frameworks.

Academic scientists sometimes tend to overlook legal or socioeco-nomic constraints, which regula-tors have to consider on a regular basis. Moreover, quantification of species abundance or biomass remains a significant bottleneck to releasing the full potential of these approaches. Here, we highlight the main challenges for next-gen-

eration aquatic biomonitoring and outline principles and good practices to address these with an emphasis on bridging traditional disciplinary boundaries between academics, regulators, stakeholders and industry.

WG5 Paper

“Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive”https://doi.org/10.1016/j.watres.2018.03.003

The abstract of the paper is: Assessment of ecological status

for the European Water Frame-work Directive (WFD) is based on “Biological Quality Elements” (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identi-fication of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches.

We rate the applicability of DNA-based identification for the individual BQEs and water cat-egories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based

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identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identifi-cation (knowledge about uncer-tainty), comparability with con-ventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environ-mental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electro-fishing.

Furthermore, there are attempts to replace absolute by relative abundance in metric calculations.

For invertebrates and phytoben-thos, the main challenges include the modification of indices and completing barcode libraries.

For phytoplankton, the barcode libraries are even more problem-atic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms / macroalgae (transitional and coastal waters), which are surveyed rather than sampled.

We discuss general implications of implementing DNA-based iden-tification into standard ecological assessment, in particular consider-ing any adaptations to the WFD that may be required to facilitate the transition to molecular data.