Tourmaline: a workflow for rapid and reproducible amplicon sequence analysis using QIIME 2 and Snakemake

Background: Amplicon sequencing (metabarcoding) is a common method to survey diversity of environmental communities whereby a single genetic locus is amplified and sequenced from the DNA of whole or partial organisms, organismal traces (e.g., skin, mucus, feces), or microbes in an environmental sample. Several software packages exist for analyzing amplicon data, among which QIIME 2 has emerged as a popular option because of its broad functionality, plugin architecture, provenance tracking, and interactive visualizations. However, each new analysis requires the user to keep track of input and output file names, parameters, and commands; this lack of automation and standardization is inefficient and creates barriers to meta-analysis and sharing of results. Findings: We developed Tourmaline, a Python-based workflow for QIIME 2 built using the Snakemake workflow management system. Starting from a configuration file that defines parameters and input files--a reference database, a sample metadata file, and a manifest or archive of FASTQ sequences--it runs either the DADA2 or Deblur denoising algorithm, assigns taxonomy to the resulting representative sequences, performs analyses of taxonomic, alpha, and beta diversity, and generates an HTML report summarizing and linking to the output files. Features include automatic determination of trimming parameters using quality scores, representative sequence filtering (taxonomy, length, abundance, prevalence, or identifier), support for multiple taxonomic classification and sequence alignment methods, outlier detection, and automated initialization of a new analysis using previous settings. The workflow runs natively on Linux and macOS or via a Docker container. We ran Tourmaline on a 16S rRNA amplicon dataset from Lake Erie surface water, showing its utility for parameter optimization and the ability to easily evaluate results through the HTML report, QIIME 2 viewer, and R- and Python-based Jupyter notebooks. Conclusions: Reproducible workflows like Tourmaline enable rapid analysis of environmental and biomedical amplicon data, decreasing the time from data generation to actionable results. Tourmaline is available for download at github.com/aomlomics/tourmaline.

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UNBIAS: An attempt to correct abundance bias in 16S sequencing, with limited success

10.1101/124149 ◽

2017 ◽

Cited By ~ 23

Author(s):

Robert C. Edgar

Keyword(s):

Copy Number ◽

Species Abundance ◽

Gene Copy Number ◽

Amplicon Sequencing ◽

Gene Copy ◽

Reference Database ◽

Abundance Distribution ◽

Frequency Distributions ◽

16S Sequencing ◽

Alpha And Beta Diversity

AbstractNext-generation amplicon sequencing of 16S ribosomal RNA is widely used to survey microbial communities. Alpha and beta diversities of these communities are often quantified on the basis of OTU frequencies in the reads. Read abundances are biased by factors including 16S copy number and PCR primer mismatches which can cause the read abundance distribution to diverge substantially from the species abundance distribution. Using mock community tests with species abundances determined independently by shotgun sequencing, I find that 16S amplicon read frequencies have no meaningful correlation with species frequencies (Pearson coefficientrclose to zero). In addition, I show that that the Jaccard distance between the abundance distributions for reads of replicate samples, which ideally would be zero, is typically ~0.15 with values up to 0.71 for replicates sequenced in different runs. Using simulated communities, I estimate that the average rank of a dominant species in the reads is 3. I describe UNBIAS, a method that attempts to correct for abundance bias due to gene copy number and primer mismatches. I show that UNBIAS can achieve informative, but still poor, correlations (r~0.6) between estimated and true abundances in the idealized case of mock samples where species are well known. However,rfalls to ~0.4 when the closest reference species have 97% identity and to ~0.2 at 95% identity. This degradation is mostly explained by the increased difficulty in predicting 16S copy number when OTUs have lower similarity with the reference database, as will typically be the case in practice. 16S abundance bias therefore remains an unsolved problem, calling into question the naive use of alpha and beta diversity metrics based on frequency distributions.

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Retinitis pigmentosa is associated with shifts in the gut microbiome

Scientific Reports ◽

10.1038/s41598-021-86052-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Oksana Kutsyr ◽

Lucía Maestre-Carballa ◽

Mónica Lluesma-Gomez ◽

Manuel Martinez-Garcia ◽

Nicolás Cuenca ◽

...

Keyword(s):

Retinitis Pigmentosa ◽

Gut Microbiome ◽

Functional Decline ◽

Retinal Disease ◽

Photoreceptor Cells ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Sequencing Data ◽

Alpha And Beta Diversity ◽

Potential Biomarker

AbstractThe gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP). Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice. In rd10 mice, retinal responsiveness to flashlight stimuli and visual acuity were deteriorated with respect to observed in age-matched control mice. This functional decline in dystrophic animals was accompanied by photoreceptor loss, morphologic anomalies in photoreceptor cells and retinal reactive gliosis. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. Remarkably, four fairly common ASV in healthy gut microbiome belonging to Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp. were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with RP. The results indicate that retinal degenerative changes in RP are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.

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Associations Between Obesity and Alzheimer’s Disease: Multiple Bioinformatic Analyses

Journal of Alzheimer s Disease ◽

10.3233/jad-201235 ◽

2021 ◽

pp. 1-11

Author(s):

Qi-Shuai Zhuang ◽

Lei Meng ◽

Zhe Wang ◽

Liang Shen ◽

Hong-Fang Ji

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Network Analysis ◽

Drug Targets ◽

Protective Factor ◽

Genetic Locus ◽

Meta Analysis ◽

Current Evidence ◽

Causal Association ◽

Ppi Network

Background: Identifying modifiable risk factors, such as obesity, to lower the prevalence of Alzheimer’s disease (AD) has gained much interest. However, whether the association is causal remains to be evaluated. Objective: The present study was designed: 1) to make a quantitative assessment of the association between obesity and AD; 2) to validate whether there was a causal association between them; and 3) to provide genetic clues for the association through a network-based analysis. Methods: Two-sample Mendelian randomization (2SMR) analysis, meta-analysis, and protein-protein interaction (PPI) network analysis, were employed. Results: Firstly, the meta-analysis based on 9 studies comprising 6,986,436 subjects indicated that midlife obesity had 33%higher AD odds than controls (OR = 1.33, 95%CI = [1.03, 1.62]), while late-life obesity were inversely associated with AD risk (OR = 0.57, 95%CI = [0.47, 0.68]). Secondly, 2SMR analysis indicated that there was no causal association between them. Thirdly, CARTPT was identified to be shared by the anti-obesity drug targets and AD susceptibility genes. Further PPI network analysis found that CARTPT interacted with CD33, a strong genetic locus linked to AD. Finally, 2SMR analysis showed that CNR1 could be a protective factor for AD. Conclusion: Multiple bioinformatic analyses indicated that midlife obesity might increase the risk of AD, while current evidence indicated that there was no causal association between them. Further, CARTPT might be an important factor linking the two disease conditions. It could help to better understand the mechanisms underlying the associations between obesity and AD.

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Reporting standards for literature searches and report inclusion criteria: Making research syntheses more transparent and easy to replicate

10.31234/osf.io/fj34p ◽

2016 ◽

Author(s):

Hannah Moshontz

Keyword(s):

Literature Search ◽

Meta Analysis ◽

Research Synthesis ◽

Search Strategies ◽

Reference Database ◽

Synthesis Methods ◽

Reporting Standards ◽

Inclusion Criteria ◽

Literature Searches ◽

Made In

A complete description of the literature search, including the criteria used for the inclusion of reports after they have been located, used in a research synthesis or meta-analysis is critical if subsequent researchers are to accurately evaluate and reproduce a synthesis' methods and results. Based on previous guidelines and new suggestions, we present a set of focused and detailed standards for reporting the methods used in a literature search. The guidelines cover five search strategies: reference database searches, journal and bibliography searches, searches of the reference lists of reports, citation searches, and direct contact searches. First, we bring together all the unique recommendations made in existing guidelines for research synthesis. Second, we identify gaps in reporting standards for search strategies. Third, we address these gaps by providing new reporting recommendations. Our hope is to facilitate successful evaluation and replication of research synthesis results.

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Prospective evaluation of the fecal microbiome in dogs with large-cell lymphoma receiving chop chemotherapy

10.32469/10355/88084 ◽

2021 ◽

Author(s):

◽

Jason Couto

Keyword(s):

Microbial Diversity ◽

Beta Diversity ◽

Large Cell ◽

Amplicon Sequencing ◽

Healthy Controls ◽

Chop Chemotherapy ◽

Fecal Samples ◽

Fecal Microbiome ◽

Alpha And Beta Diversity ◽

Healthy Dogs

The fecal microbiome composition has been associated with reduced efficacy of cancer therapy and adverse side effects in humans, and chemotherapy has been shown to alter the gut microbiome. The relationship between microbiota and chemotherapy efficacy and tolerability has not been investigated in dogs. We aimed to evaluate changes in fecal microbial diversity during a cycle of CHOP chemotherapy in dogs with lymphoma and whether these changes correlated with adverse events or treatment response. Eighteen dogs with lymphoma were prospectively enrolled, and stool samples were acquired weekly for 6 weeks during CHOP. Fecal samples was analyzed via 16S rRNA amplicon sequencing as previously described. Treatment-associated differences in richness, alpha and beta diversity were determined through comparison to data from healthy controls (n = 26) using factorial ANOVA and PERMANOVA. Dogs with lymphoma had decreased fecal microbial diversity when compared with healthy controls at baseline and throughout treatment (p= 0.0002, 0.0003, 0.0001). Alpha and beta diversity did not significantly change in dogs throughout a cycle of CHOP chemotherapy (p = 0.520 and 0.995). Samples pre-treated with antibiotics were significantly less diverse (alpha and beta diversity) than untreated samples (p = 0.002, 0.0001 respectively). Dogs with lymphoma and fecal samples under the presence of antibiotics had higher levels of Escherchia species in their feces compared to normal dogs. The fecal microbiome of healthy dogs and dogs with lymphoma receiving CHOP is relatively stable over time, but dogs with lymphoma have reduced microbial diversity compared to healthy dogs before and during treatment. An increase in Proteobacteria abundance during treatment may be related to chemotherapy and/or antibiotic use.

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Natrix: a Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads

BMC Bioinformatics ◽

10.1186/s12859-020-03852-4 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Marius Welzel ◽

Anja Lange ◽

Dominik Heider ◽

Michael Schwarz ◽

Bernd Freisleben ◽

...

Keyword(s):

High Throughput Sequencing ◽

Workflow Management ◽

Amplicon Sequencing ◽

Version Control ◽

Marker Genes ◽

Sequencing Data ◽

Taxonomic Assignment ◽

Ecological Processes ◽

Link Type ◽

User Friendly

Abstract Background Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. Results We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub (https://github.com/MW55/Natrix) or as a Docker container on DockerHub (https://hub.docker.com/r/mw55/natrix). Conclusion Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

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Meta-Analysis of Microbial Communities in Hot Springs: Recurrent Taxa and Complex Shaping Factors beyond pH and Temperature

Microorganisms ◽

10.3390/microorganisms8060906 ◽

2020 ◽

Vol 8 (6) ◽

pp. 906 ◽

Cited By ~ 2

Author(s):

Francisco L. Massello ◽

Chia Sing Chan ◽

Kok-Gan Chan ◽

Kian Mau Goh ◽

Edgardo Donati ◽

...

Keyword(s):

Microbial Communities ◽

Metabolic Profile ◽

Hot Springs ◽

Meta Analysis ◽

Extreme Environments ◽

Alpha Diversity ◽

Amplicon Sequencing ◽

Phylogenetic Distance ◽

Microbial Profile ◽

The World

The study of microbial communities from extreme environments is a fascinating topic. With every study, biologists and ecologists reveal interesting facts and questions that dispel the old belief that these are inhospitable environments. In this work, we assess the microbial diversity of three hot springs from Neuquén, Argentina, using high-throughput amplicon sequencing. We predicted a distinct metabolic profile in the acidic and the circumneutral samples, with the first ones being dominated by chemolithotrophs and the second ones by chemoheterotrophs. Then, we collected data of the microbial communities of hot springs around the world in an effort to comprehend the roles of pH and temperature as shaping factors. Interestingly, there was a covariation between both parameters and the phylogenetic distance between communities; however, neither of them could explain much of the microbial profile in an ordination model. Moreover, there was no correlation between alpha diversity and these parameters. Therefore, the microbial communities’ profile seemed to have complex shaping factors beyond pH and temperature. Lastly, we looked for taxa associated with different environmental conditions. Several such taxa were found. For example, Hydrogenobaculum was frequently present in acidic springs, as was the Sulfolobaceae family; on the other hand, Candidatus Hydrothermae phylum was strongly associated with circumneutral conditions. Interestingly, some singularities related to sites featuring certain taxa were also observed.

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Gut microbiota composition is associated with narcolepsy type 1

Neurology Neuroimmunology & Neuroinflammation ◽

10.1212/nxi.0000000000000896 ◽

2020 ◽

Vol 7 (6) ◽

pp. e896

Author(s):

Alexandre Lecomte ◽

Lucie Barateau ◽

Pedro Pereira ◽

Lars Paulin ◽

Petri Auvinen ◽

...

Keyword(s):

Community Structure ◽

Gut Microbiota ◽

Bacterial Communities ◽

Beta Diversity ◽

Alpha Diversity ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Differential Abundance ◽

Alpha And Beta Diversity

ObjectiveTo test the hypothesis that narcolepsy type 1 (NT1) is related to the gut microbiota, we compared the microbiota bacterial communities of patients with NT1 and control subjects.MethodsThirty-five patients with NT1 (51.43% women, mean age 38.29 ± 19.98 years) and 41 controls (57.14% women, mean age 36.14 ± 12.68 years) were included. Stool samples were collected, and the fecal microbiota bacterial communities were compared between patients and controls using the well-standardized 16S rRNA gene amplicon sequencing approach. We studied alpha and beta diversity and differential abundance analysis between patients and controls, and between subgroups of patients with NT1.ResultsWe found no between-group differences for alpha diversity, but we discovered in NT1 a link with NT1 disease duration. We highlighted differences in the global bacterial community structure as assessed by beta diversity metrics even after adjustments for potential confounders as body mass index (BMI), often increased in NT1. Our results revealed differential abundance of several operational taxonomic units within Bacteroidetes, Bacteroides, and Flavonifractor between patients and controls, but not after adjusting for BMI.ConclusionWe provide evidence of gut microbial community structure alterations in NT1. However, further larger and longitudinal multiomics studies are required to replicate and elucidate the relationship between the gut microbiota, immunity dysregulation and NT1.

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PSVI-16 Fecal microbiome of nursery pigs fed phytogenics and antibiotics

Journal of Animal Science ◽

10.1093/jas/skz258.418 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 203-203

Author(s):

Huyen Tran ◽

Timothy J Johnson

Keyword(s):

Beta Diversity ◽

Sequence Data ◽

Alpha Diversity ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Fecal Microbiome ◽

Nursery Pigs ◽

Alpha And Beta Diversity ◽

Family Level ◽

Dietary Treatments

Abstract The objective of this study was to evaluate effects of feeding two phytogenic products (PHY1 and PHY2; blends of essential oils and plant extracts) in diets with or without antibiotics (AureoMix S 10-10; AB) on fecal microbiome of nursery pigs. A total of 400 nursery pigs (6.8 kg BW; 20 d of age) were fed one of the six dietary treatments (9 pens/treatment), including: control (0% AB; 0% phytogenics), 0.5% AB, phytogenics (0.02% PHY1 or 0.03% PHY2) or the combination of phytogenic and AB (PHY1 x AB or PHY2 x AB). On d 46 postweaning, 48 fecal samples were collected (1 pig/pen; 7–9 pigs/treatment) and were subjected to the analyses of microbial communities by using 16S rRNA V4 amplicon sequencing with Illumina MiSeq. The sequence data were analyzed by using Qiime and the rarefied OTU table was submitted to Calypso to evaluate the alpha and beta diversity, taxonomic classification, and the differential taxa associated to the dietary treatments. There were differences among treatments on alpha diversity, where the control and PHY2 pigs had lower OTU richness (P = 0.05) and chao1 index (P < 0.10) compared to pigs fed AB alone or AB with phytogenics. There were also differences among treatments on microbial beta diversity of pigs (P < 0.01). The most abundant phyla included Firmicute, Bacteroidetes, Actinobacteria, Tenericutes, Proteobacteria, Spirochaetes, and TM7. At family level, pigs fed AB had greater Ruminococcaceae compared to the control, but lower Coriobacteriaceae and Erysipelotrichaceae compared to PHY1 or PHY2 group (P < 0.05). Feature selection by LEfSe indicated that dominant genus associated to AB treatment was Unclassified RF39, while dominant genera associated to PHY2 treatment were Cantenibacterium, unclassified Coriobacteriaceae, Blautia, Eubacterium, and Collinsella. In conclusion, feeding AB and phytogenic products had different impacts on the fecal bacteria of nursery pigs.

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An association between chronic widespread pain and the gut microbiome

Rheumatology ◽

10.1093/rheumatology/keaa847 ◽

2020 ◽

Author(s):

Maxim B Freidin ◽

Maria A Stalteri ◽

Philippa M Wells ◽

Genevieve Lachance ◽

Andrei-Florin Baleanu ◽

...

Keyword(s):

Gut Microbiome ◽

Genome Wide Association Study ◽

Meta Analysis ◽

Alpha Diversity ◽

Amplicon Sequencing ◽

Chronic Widespread Pain ◽

Mendelian Randomisation ◽

Total N ◽

16S Rrna Amplicon Sequencing ◽

A Genome

Abstract Objectives Chronic widespread musculoskeletal pain (CWP) is a characteristic symptom of fibromyalgia, which has been shown to be associated with an altered gut microbiome. Microbiome studies to date have not examined the milder CWP phenotype specifically nor have they explored the role of raised BMI. The aim of this study was to investigate whether the microbiome is abnormal in CWP. Methods CWP was assessed using a standardized screening questionnaire in female volunteers from the TwinsUK cohort including 113 CWP cases and 1623 controls. The stool microbiome was characterised using 16S rRNA amplicon sequencing and amplicon sequence variants (ASVs), and associations with CWP examined using linear mixed-effects models adjusting for BMI, age, diet, family relatedness and technical factors. Results Alpha diversity was significantly lower in CWP cases than controls (Mann–Whitney test, p-values 2.3e-04 and 1.2e-02, respectively). The species Coprococcus comes was significantly depleted in CWP cases (p.adj = 3.04e-03). A genome-wide association study (GWAS) performed for C. comes in TwinsUK followed by meta-analysis with three Dutch cohorts (total n = 3521) resulted in nine suggestive regions, with the most convincing on chromosome 4 near the TRAM1L1 gene (rs76957229, p= 7.4e-8). A Mendelian randomisation study based on the results of the GWAS did not support a causal role for C. comes on the development of CWP. Conclusions We have demonstrated reduced diversity in the microbiome in CWP, indicating an involvement of the gut microbiota in CWP; prospectively the microbiome may offer therapeutic opportunities for this condition.

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