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      Freshwater diatom biomonitoring through benthic kick-net metabarcoding

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          Abstract

          Biomonitoring is an essential tool for assessing ecological conditions and informing management strategies. The application of DNA metabarcoding and high throughput sequencing has improved data quantity and resolution for biomonitoring of taxa such as macroinvertebrates, yet, there remains the need to optimise these methods for other taxonomic groups. Diatoms have a longstanding history in freshwater biomonitoring as bioindicators of water quality status. However, multi-substrate periphyton collection, a common diatom sampling practice, is time-consuming and thus costly in terms of labour. This study examined whether the benthic kick-net technique used for macroinvertebrate biomonitoring could be applied to bulk-sample diatoms for metabarcoding. To test this approach, we collected samples using both conventional multi-substrate microhabitat periphyton collections and bulk-tissue kick-net methodologies in parallel from replicated sites with different habitat status (good/fair). We found there was no significant difference in community assemblages between conventional periphyton collection and kick-net methodologies or site status, but there was significant difference between diatom communities depending on site ( P = 0.042). These results show the diatom taxonomic coverage achieved through DNA metabarcoding of kick-net is suitable for ecological biomonitoring applications. The shift to a more robust sampling approach and capturing diatoms as well as macroinvertebrates in a single sampling event has the potential to significantly improve efficiency of biomonitoring programmes that currently only use the kick-net technique to sample macroinvertebrates.

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          Cutadapt removes adapter sequences from high-throughput sequencing reads

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            Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.

            The Ribosomal Database Project (RDP) Classifier, a naïve Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (> or = 95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/.
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              Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness

              Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 November 2020
                2020
                : 15
                : 11
                : e0242143
                Affiliations
                [001]Centre for Biodiversity Genomics & Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada
                University of Hyogo, JAPAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0003-3776-9166
                https://orcid.org/0000-0002-0227-6874
                Article
                PONE-D-20-15884
                10.1371/journal.pone.0242143
                7673570
                33206700
                e264342e-8d13-4bed-92a4-42f7c16d7da5
                © 2020 Maitland et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 May 2020
                : 27 October 2020
                Page count
                Figures: 7, Tables: 0, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100008762, Genome Canada;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000159, Natural Resources Canada;
                Award Recipient :
                This study is funded by the Government of Canada through Genome Canada and Ontario Genomics. Teresita Porter was funded by the Government of Canada through the Genomics Research and Development Initiative (GRDI) Metagenomics-Based Ecosystem Biomonitoring Project (Ecobiomics).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Algae
                Phytoplankton
                Diatoms
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Plankton
                Phytoplankton
                Diatoms
                Biology and Life Sciences
                Zoology
                Animals
                Invertebrates
                Plankton
                Phytoplankton
                Diatoms
                Biology and Life Sciences
                Taxonomy
                Computer and Information Sciences
                Data Management
                Taxonomy
                Ecology and Environmental Sciences
                Aquatic Environments
                Freshwater Environments
                Fresh Water
                Earth Sciences
                Marine and Aquatic Sciences
                Aquatic Environments
                Freshwater Environments
                Fresh Water
                Earth Sciences
                Geology
                Petrology
                Sediment
                Earth Sciences
                Geology
                Sedimentary Geology
                Sediment
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Plankton
                Biology and Life Sciences
                Zoology
                Animals
                Invertebrates
                Plankton
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Mechanical Treatment of Specimens
                Specimen Scraping
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Ecology
                Community Ecology
                Community Assembly
                Ecology and Environmental Sciences
                Ecology
                Community Ecology
                Community Assembly
                Custom metadata
                All raw sequence files are available from the NCBI SRA BioProject database (accession number PRJNA673880).

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                Uncategorized

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