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In October of 2020, in response to the Coronavirus Disease 2019 (COVID-19) pandemic, our team hosted our first fully online workshop teaching the QIIME 2 microbiome bioinformatics platform. We had 75 enrolled participants who joined from at least 25 different countries on 6 continents, and we had 22 instructors on 4 continents. In the 5-day workshop, participants worked hands-on with a cloud-based shared compute cluster that we deployed for this course. The event was well received, and participants provided feedback and suggestions in a postworkshop questionnaire. In January of 2021, we followed this workshop with a second fully online workshop, incorporating lessons from the first. Here, we present details on the technology and protocols that we used to run these workshops, focusing on the first workshop and then introducing changes made for the second workshop. We discuss what worked well, what didn’t work well, and what we plan to do differently in future workshops., PLoS Computational Biology, 17 (6), ISSN:1553-734X, ISSN:1553-7358

BackgroundNucleotide sequence and taxonomy reference databases are critical resources for widespread applications including marker-gene and metagenome sequencing for microbiome analysis, diet metabarcoding, and environmental DNA (eDNA) surveys. Reproducibly generating, managing, using, and evaluating nucleotide sequence and taxonomy reference databases creates a significant bottleneck for researchers aiming to generate custom sequence databases. Furthermore, database composition drastically influences results, and lack of standardizations limits cross-study comparisons. To address these challenges, we developed RESCRIPt, a software package for reproducible generation and management of reference sequence taxonomy databases, including dedicated functions that streamline creating databases from popular sources, and functions for evaluating, comparing, and interactively exploring qualitative and quantitative characteristics across reference databases.ResultsTo highlight the breadth and capabilities of RESCRIPt, we provide several examples for working with popular databases for microbiome profiling (SILVA, Greengenes, NCBI-RefSeq, GTDB), eDNA, and diet metabarcoding surveys (BOLD, GenBank), as well as for genome comparison. We show that bigger is not always better, and reference databases with standardized taxonomies and those that focus on type strains have quantitative advantages, though may not be appropriate for all use cases. Most databases appear to benefit from some curation (quality filtering), though sequence clustering appears detrimental to database quality. Finally, we demonstrate the breadth and extensibility of RESCRIPt for reproducible workflows with a comparison of global hepatitis genomes.ConclusionsRESCRIPt provides tools to democratize the process of reference database acquisition and management, enabling researchers to reproducibly and transparently create reference materials for diverse research applications. RESCRIPt is released under a permissive BSD-3 license athttps://github.com/bokulich-lab/RESCRIPt.

The COVID-19 pandemic has led to a rapid accumulation of SARS-CoV-2 genomes, enabling genomic epidemiology on local and global scales. Collections of genomes from resources such as GISAID must be subsampled to enable computationally feasible phylogenetic and other analyses. We present genome-sampler, a software package that supports sampling collections of viral genomes across multiple axes including time of genome isolation, location of genome isolation, and viral diversity. The software is modular in design so that these or future sampling approaches can be applied independently and combined (or replaced with a random sampling approach) to facilitate custom workflows and benchmarking. genome-sampler is written as a QIIME 2 plugin, ensuring that its application is fully reproducible through QIIME 2’s unique retrospective data provenance tracking system. genome-sampler can be installed in a conda environment on macOS or Linux systems. A complete default pipeline is available through a Snakemake workflow, so subsampling can be achieved using a single command. genome-sampler is open source, free for all to use, and available at https://caporasolab.us/genome-sampler. We hope that this will facilitate SARS-CoV-2 research and support evaluation of viral genome sampling approaches for genomic epidemiology., F1000Research, 9, ISSN:2046-1402

QIIME 2 is a platform for microbiome bioinformatics that serves researchers around the world. Microbiomes are communities of microorganisms that are ubiquitous in our bodies and on our planet. Advances in DNA sequencing and bioinformatics technologies in the past few decades have provided a new lens into the microbial world, and developing our understanding of microbiomes promises to improve diverse aspects of human health, assist with environmental sustainability and feeding of 8 billion people, and maybe even save us from a post-antibiotic world. The QIIME project has existed for just over 10 years, and has been cited in nearly 20,000 microbiome research studies. QIIME 2 succeeded QIIME 1 on Jan 1 2018. QIIME 2 is a plugin-based platform that enables reproducible microbiome research through different interface types.

This study offers a novel description of the sinonasal microbiome, through an unsupervised machine learning approach combining dimensionality reduction and clustering. We apply our method to the International Sinonasal Microbiome Study (ISMS) dataset of 410 sinus swab samples. We propose three main sinonasal “microbiotypes” or “states”: the first is Corynebacterium-dominated, the second is Staphylococcus-dominated, and the third dominated by the other core genera of the sinonasal microbiome (Streptococcus, Haemophilus, Moraxella, and Pseudomonas). The prevalence of the three microbiotypes studied did not differ between healthy and diseased sinuses, but differences in their distribution were evident based on geography. We also describe a potential reciprocal relationship between Corynebacterium species and Staphylococcus aureus, suggesting that a certain microbial equilibrium between various players is reached in the sinuses. We validate our approach by applying it to a separate 16S rRNA gene sequence dataset of 97 sinus swabs from a different patient cohort. Sinonasal microbiotyping may prove useful in reducing the complexity of describing sinonasal microbiota. It may drive future studies aimed at modeling microbial interactions in the sinuses and in doing so may facilitate the development of a tailored patient-specific approach to the treatment of sinus disease in the future.

QIIME 2 is a completely re‐engineered microbiome bioinformatics platform based on the popular QIIME platform, which it has replaced. QIIME 2 facilitates comprehensive and fully reproducible microbiome data science, improving accessibility to diverse users by adding multiple user interfaces. QIIME 2 can be combined with Qiita, an open‐source web‐based platform, to re‐use available data for meta‐analysis. The following basic protocol describes how to install QIIME 2 on a single computer and analyze microbiome sequence data, from processing of raw DNA sequence reads through generating publishable interactive figures. These interactive figures allow readers of a study to interact with data with the same ease as its authors, advancing microbiome science transparency and reproducibility. We also show how plug‐ins developed by the community to add analysis capabilities can be installed and used with QIIME 2, enhancing various aspects of microbiome analyses—e.g., improving taxonomic classification accuracy. Finally, we illustrate how users can perform meta‐analyses combining different datasets using readily available public data through Qiita. In this tutorial, we analyze a subset of the Early Childhood Antibiotics and the Microbiome (ECAM) study, which tracked the microbiome composition and development of 43 infants in the United States from birth to 2 years of age, identifying microbiome associations with antibiotic exposure, delivery mode, and diet. For more information about QIIME 2, see https://qiime2.org. To troubleshoot or ask questions about QIIME 2 and microbiome analysis, join the active community at https://forum.qiime2.org., Current protocols in bioinformatics, 70 (1)

Multi-omic insights into microbiome function and composition typically advance one study at a time. However, in order for relationships across studies to be fully understood, data must be aggregated into meta-analyses. This makes it possible to generate new hypotheses by finding features that are reproducible across biospecimens and data layers. Qiita dramatically accelerates such integration tasks in a web-based microbiome-comparison platform, which we demonstrate with Human Microbiome Project and Integrative Human Microbiome Project (iHMP) data.

This study offers a novel description of the sinonasal microbiome, through an unsupervised machine learning approach combining dimensionality reduction and clustering. We apply our method to the International Sinonasal Microbiome Study (ISMS) dataset of 410 sinus swab samples. We propose three main sinonasal ‘microbiotypes’ or ‘states’: the first is Corynebacterium-dominated, the second is Staphylococcus-dominated, and the third dominated by the other core genera of the sinonasal microbiome (Streptococcus, Haemophilus, Moraxella, and Pseudomonas). The prevalence of the three microbiotypes studied did not differ between healthy and diseased sinuses, but differences in their distribution were evident based on geography. We also describe a potential reciprocal relationship between Corynebacterium species and Staphylococcus aureus, suggesting that a certain microbial equilibrium between various players is reached in the sinuses. We validate our approach by applying it to a separate 16S rRNA gene sequence dataset of 97 sinus swabs from a different patient cohort. Sinonasal microbiotyping may prove useful in reducing the complexity of describing sinonasal microbiota. It may drive future studies aimed at modeling microbial interactions in the sinuses and in doing so may facilitate the development of a tailored patient-specific approach to the treatment of sinus disease in the future.

The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical divides remains unexplored. We characterise the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 individuals from across the world. Although the sinus microbial ecology is highly variable between individuals, we identify a core microbiome comprised of Corynebacterium, Staphylococcus, Streptococcus, Haemophilus, and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients sampled. There was a significant variation in microbial diversity between countries (p = 0.001). Amongst patients suffering from CRS with nasal polyps, a significant depletion of Corynebacterium (40.29% vs 50.43%; p = 0.02) and over-representation of Streptococcus (7.21% vs 2.73%; p = 0.032) was identified. The delineation of the sinonasal microbiome and standardised methodology described within our study will enable further characterisation and translational application of the sinus microbiota.

QIIME 2 development was primarily funded by NSF Awards 1565100 to J.G.C. and 1565057 to R.K. Partial support was also provided by the following: grants NIH U54CA143925 (J.G.C. and T.P.) and U54MD012388 (J.G.C. and T.P.); grants from the Alfred P. Sloan Foundation (J.G.C. and R.K.); ERCSTG project MetaPG (N.S.); the Strategic Priority Research Program of the Chinese Academy of Sciences QYZDB-SSW-SMC021 (Y.B.); the Australian National Health and Medical Research Council APP1085372 (G.A.H., J.G.C., Von Bing Yap and R.K.); the Natural Sciences and Engineering Research Council (NSERC) to D.L.G.; and the State of Arizona Technology and Research Initiative Fund (TRIF), administered by the Arizona Board of Regents, through Northern Arizona University. All NCI coauthors were supported by the Intramural Research Program of the National Cancer Institute. S.M.G. and C. Diener were supported by the Washington Research Foundation Distinguished Investigator Award.

We present QIIME 2, an open-source microbiome data science platform accessible to users spanning the microbiome research ecosystem, from scientists and engineers to clinicians and policy makers. QIIME 2 provides new features that will drive the next generation of microbiome research. These include interactive spatial and temporal analysis and visualization tools, support for metabolomics and shotgun metagenomics analysis, and automated data provenance tracking to ensure reproducible, transparent microbiome data science.

AnIntroduction toAppliedBioinformatics (or IAB) is afree, open source interactive textthat introduces readers to core concepts of bioinformatics in the context of their implementation and application. The archived version corresponds to the master branch of the GitHub repository at the time of acceptance of a paper describing this work in the Journal of Open Source Education.

The Journal of Open Source Software, 3 (30), ISSN:2475-9066

Statement of need: Due to the increasing rate of biological data generation, bioinformatics is rapidly growing as a field and is now an essential part of scientific advances in human health and environmental sciences. Online and publicly accessible resources for learning bioinformatics exist (e.g., Rosalind, (Searls, 2012, 2014)), and there are excellent textbooks and courses in the area, some focused heavily on theory (Durbin, Eddy, Krogh, & Mitchison, 1998; Felsenstein, 2003), and others geared toward learning specific skills such as Python programming or the Unix shell (Dunn & Haddock, 2010; Wilson, 2016). An Introduction to Applied Bioinformatics (IAB) is a free, online bioinformatics text that bridges the gap between theory and application by teaching fundamentals of bioinformatics in the context of their implementation, using an interactive framework based on highly relevant tools including Python 3, Jupyter Notebooks, and GitHub. IAB is geared toward students who are completely new to bioinformatics, though having completed an introductory course (or book) in both Computer Science and Biology are useful prerequisites. IAB readers begin on the project website. While it is possible to view the content statically from this page, we recommend that readers work interactively by installing IAB. Readers progress through chapters that introduce fundamental topics, such as sequence homology searching and multiple sequence alignment, and presents their Python 3 implementation. Because the content is presented in Jupyter Notebooks, students can edit and execute the code, for example to explore how changing k-word size or an alignment gap penalty might impact the results of a database search. The Python code that readers interact with is intended for educational purposes, where the implementation is made as simple as possible, sometimes at the cost of computational efficiency. Chapters therefore also include examples of performing the same analyses with scikit-bio, a production-quality bioinformatics Python 3 library. This enables a rapid transition from learning theory, or how an algorithm works, to applying techniques in a real-world setting. IAB additionally contains Wikipedia-style “Edit” links in each section of the text. When one of these links is followed, the reader is taken to the GitHub online editor where they can submit a pull request to modify content or code. Readers are therefore introduced to GitHub through a user-friendly web interface, and can begin building their GitHub activity history (commonly reviewed by bioinformatics hiring managers). Finally, every time a change is proposed via GitHub, all of the executable content of IAB is automatically tested. This continuous integration testing ensures that IAB example code remains functional as changes are introduced, solving an issue that plagues printed applied computational texts (for example because they describe an outdated software interface). IAB evolved from lecture materials developed by Dr. Caporaso for an introductory bioinformatics course targeted toward computer science and biology undergraduates (typically juniors or seniors) at Northern Arizona University. Since the early stages of its development, it has been used to teach at least ten courses and short (e.g., one day) bioinformatics workshops. As it became clear that the content and format was useful for teaching bioinformatics, Dr. Caporaso applied for and received grants from the Arizona Technology and Research Initiative and the Alfred P Sloan Foundation to further develop the resource.The content was originally written in Jupyter Notebooks, but as the project grew, it became difficult to maintain the notebooks and in particular to review submissions from others. The Jupyter Notebooks were transitioned to markdown files which are now the source for static HTML and Jupyter Notebook renderings of the content. The current version of IAB contains six chapters covering fundamental concepts and their applications. It is a dynamic resource that will be expanded, revised and updated over time. Its lifecycle is thus more similar to an active software project than a textbook: a practical approach to education in a rapidly changing field.

Background: Taxonomic classification of marker-gene sequences is an important step in microbiome analysis. Results: We present q2-feature-classifier ( https://github.com/qiime2/q2-feature-classifier ), a QIIME 2 plugin containing several novel machine-learning and alignment-based taxonomy classifiers that meet or exceed the accuracy of existing methods for marker-gene amplicon sequence classification. We evaluated and optimized several commonly used taxonomic classification methods (RDP, BLAST, UCLUST) and several new methods (a scikit-learn naive Bayes machine-learning classifier, and alignment-based taxonomy consensus methods of VSEARCH, BLAST+, and SortMeRNA) for classification of marker-gene amplicon sequence data. Conclusions: Our results illustrate the importance of parameter tuning for optimizing classifier performance, and we make recommendations regarding parameter choices for a range of standard operating conditions. q2-feature-classifier and our evaluation framework, tax-credit, are both free, open-source, BSD-licensed packages available on GitHub.

Background Taxonomic classification of marker-gene sequences is an important step in microbiome analysis. Results We present q2-feature-classifier (https://github.com/qiime2/q2-feature-classifier), a QIIME 2 plugin containing several novel machine-learning and alignment-based methods for taxonomy classification. We evaluated and optimized several commonly used classification methods implemented in QIIME 1 (RDP, BLAST, UCLUST, and SortMeRNA) and several new methods implemented in QIIME 2 (a scikit-learn naive Bayes machine-learning classifier, and alignment-based taxonomy consensus methods based on VSEARCH, and BLAST+) for classification of bacterial 16S rRNA and fungal ITS marker-gene amplicon sequence data. The naive-Bayes, BLAST+-based, and VSEARCH-based classifiers implemented in QIIME 2 meet or exceed the species-level accuracy of other commonly used methods designed for classification of marker gene sequences that were evaluated in this work. These evaluations, based on 19 mock communities and error-free sequence simulations, including classification of simulated “novel” marker-gene sequences, are available in our extensible benchmarking framework, tax-credit (https://github.com/caporaso-lab/tax-credit-data). Conclusions Our results illustrate the importance of parameter tuning for optimizing classifier performance, and we make recommendations regarding parameter choices for these classifiers under a range of standard operating conditions. q2-feature-classifier and tax-credit are both free, open-source, BSD-licensed packages available on GitHub., Microbiome, 6 (1), ISSN:2049-2618

Studies of host-associated and environmental microbiomes often incorporate longitudinal sampling or paired samples in their experimental design. Longitudinal sampling provides valuable information about temporal trends and subject/population heterogeneity, offering advantages over cross-sectional and pre/post study designs. To support the needs of microbiome researchers performing longitudinal studies, we developed q2-longitudinal, a software plugin for the QIIME 2 microbiome analysis platform (https://qiime2.org). The q2-longitudinal plugin incorporates multiple methods for analysis of longitudinal and paired-sample data, including paired differences and distances, linear mixed effects models, microbial interdependence test, first differencing, and volatility analyses. The q2-longitudinal package (https://github.com/qiime2/q2-longitudinal) is open source software released under a BSD-3-Clause license and is freely available, including for commercial use.

Background. Taxonomic classification of marker-gene sequences is an important step in microbiome analysis. Results. We present q2-feature-classifier (https://github.com/qiime2/q2-feature-classifier), a QIIME 2 plugin containing several novel machine-learning and alignment-based taxonomy classifiers that meet or exceed classification accuracy of existing methods. We evaluated and optimized several commonly used taxonomic classification methods (RDP, BLAST, BLAST+, UCLUST) and several new methods (a scikit-learn naive Bayes machine-learning classifier, and VSEARCH and SortMeRNA alignment-based methods). Conclusions. Our results illustrate the importance of parameter tuning for optimizing classifier performance, and we make explicit recommendations regarding parameter choices for a range of standard operating conditions. q2-feature-classifier and our evaluation framework, tax-credit, are both free, open-source, BSD-licensed packages available on GitHub.

In the version of this article initially published, some reference citations were incorrect. The three references to Jupyter Notebooks should have cited Kluyver et al. instead of Gonzalez et al. The reference to Qiita should have cited Gonzalez et al. instead of Schloss et al. The reference to mothur should have cited Schloss et al. instead of McMurdie & Holmes. The reference to phyloseq should have cited McMurdie & Holmes instead of Huber et al. The reference to Bioconductor should have cited Huber et al. instead of Franzosa et al. And the reference to the biobakery suite should have cited Franzosa et al. instead of Kluyver et al. The errors have been corrected in the HTML and PDF versions of the article.

Geotechnical investigations in the vicinity of Anchorage, Alaska revealed a body of ice-rich permafrost up to 12 m thick and approximately 150 m long. Mean annual air temperature in Anchorage is + 2.2 °C which is unfavorable to the existence of permafrost. However, isolated masses of relic permafrost exist at temperatures close to 0 °C protected from rapid thawing by a thick layer of peat and soils with high ice content. Frozen soils found at the study site include silty clay of glacio-lacustrine origin with numerous layers of segregated ice up to 70 cm in thickness. The average volume of visible ice is 42.5%; and total gravimetric water content is 68%. With an average permafrost thickness of 9.5 m and an average thaw strain of 40%, the thaw settlement of the surface is expected to be at least 3.8 m after degradation of permafrost, an unacceptable deformation for any engineered structure on these soils.

Ground ice in the upper permafrost of the Beaufort Sea coast of Alaska was studied from 2005 to 2008 at 65 field sites located between Point Barrow and the Canadian border. The main terrain units in the studied area include (1) the primary surface of the coastal plain; (2) drained-lake basins; (3) low foothills (yedoma); (4) deltas and tidal flats; and (5) sand dunes. Wedge ice is the main type of massive ground ice, and ice-wedge polygons occurred on nearly all land surfaces. The volumetric content of wedge ice for the area varies from 3% to 50% between various terrain units with average value of about 11% for the entire coast. The highest content of wedge ice (about 50%) is typical of yedoma terrain, which occurred in a small segment at the coast of the Camden Bay. At the primary surface of the western region of the Arctic Coastal Plain, wedge-ice content reached almost 30%, with an average value of about 14%. Slightly smaller values were estimated for the primary surface of the eastern region of the Arctic Coastal Plain and for old drained-lake basins. Other types of massive ground ice included thermokarst-cave ice, ice cores of pingos, and a rare occurrence of folded massive ice at Barter Island. The content of segregated ice in organic and mineral soils between ice wedges was very high at most of the study sites. The total average volumetric ice content (due to wedge, segregated, and pore ice) for the whole area was 77%, ranging from 43% in eolian sand to 89% in yedoma.