Microbiome Analysis via OTU and ASV-Based Pipelines—A Comparative Interpretation of Ecological Data in WWTP Systems

Jeske, Jan Torsten; Gallert, Claudia

doi:10.3390/bioengineering9040146

Cited by 26 publications

(17 citation statements)

References 54 publications

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“…Importantly, reporting different genetic units may skew conclusions because they give different impressions of biological diversity. ASVs are often preferred to OTUs because they are transferable across studies and do not require arbitrary dissimilarity thresholds (Callahan et al, 2017; Jeske & Gallert, 2022). In addition, OTU clustering is based on haplotype similarity, so OTUs are not recommended for eNA studies that analyze intraspecific genetic diversity (Tsuji et al, 2020).…”

Section: Resultsmentioning

confidence: 99%

Environmental DNA and RNA in aquatic community ecology: Toward methodological standardization

Bunholi,

Foster,

Casey

2023

Environmental DNA

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Molecular technologies have facilitated the expansion of biodiversity assessments across a broad range of organisms and aquatic systems. Environmental DNA (eDNA) and environmental RNA (eRNA), collectively referred to as environmental nucleic acids (eNAs), have revolutionized biodiversity monitoring due to their noninvasive nature and high‐resolution capabilities when compared to traditional survey methods. While eNA applications have grown exponentially over the past decade, methodological inconsistencies hinder reproducibility and comparability. To assess the current state of eNA methodologies in aquatic ecology, we conducted a systematic review of 300 peer‐reviewed eNA studies that assess ecological communities across diverse aquatic systems. Of these papers, 291 examine eDNA, four examine eRNA, and five consider both eDNA and eRNA. The small number of eRNA studies compared to eDNA highlights the eRNA field is in its infancy. Of the eNA studies looked at, we found a clear geographic bias, with approximately 50% of all eNA studies occurring in six high‐income countries, while less than 10% of eNA studies occur across South America and Africa. Further, we report a lack of methodological standardization across eNA studies, showing high variability in water filtration volume, filter material, filter pore size, eNA extraction method, marker choice, and bioinformatic pipelines. We further highlight that incomplete reference sequence databases for both eRNA and eDNA limit taxonomic assignment and biodiversity inferences. Finally, we identify a systematic issue across eNA studies in community ecology: missing methodological details, which compromise reproducibility, especially in newly emerging eRNA applications. To facilitate the standardization of eNA monitoring across aquatic systems and permit the further integration of eNA applications in biodiversity monitoring, we recommend the improvement of reference databases alongside guidelines that encourage methodological transparency.

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Section: Resultsmentioning

confidence: 99%

Environmental DNA and RNA in aquatic community ecology: Toward methodological standardization

Bunholi,

Foster,

Casey

2023

Environmental DNA

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“…Studies that rely on ASVs use statistical models intended to remove the errors associated with sequencing, returning individual and unique sequences that represent individual taxa (Kerrigan & D'Hondt, 2022; Prodan et al., 2020). Therefore, ASV approaches can provide a significant advantage since a single base difference in the sequence will result in a unique ASV, and a more precise identification of the diversity of a given sample (Jeske & Gallert, 2022).…”

Section: Methodsmentioning

confidence: 99%

Geomicrobiology of the Rincon de Parangueo maar crater: Exploring the link between an evolving extreme environment and its potential metabolic diversity

Sánchez‐Sánchez,

Cerca,

Moguel

et al. 2023

Freshwater Biology

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Rincon de Parangueo (RP) is a Quaternary maar crater located in the Michoacan‐Guanajuato Volcanic Field in central Mexico. Like other volcanic craters in the region, the central part was occupied by an endorheic lacustrine system. As a consequence of extensive groundwater extraction, the perennial lake started a gradual desiccation process and now the remnant ponds host a highly saline–alkaline ecosystem. The stratigraphic record indicates an exceptional and long‐term sedimentation of carbonate microbialites. Previous studies based on the 16S rRNA gene have shown that microbial communities have a widespread presence in the crater at the microbialites, the remaining saline ponds, superficial soils and below the surface. To understand the possible role of the microbial communities that generate these biogeological structures, novel analytical methods to resolve the amplicon datasets and their microbial metabolic potential were used. We describe in detail the relationship between the microbial communities, the evolution of physicochemical parameters, and carbonate morphology, in four micro‐environments. A 16S rRNA gene sequencing dataset from previous publications was re‐analysed using an ASV‐based bioinformatic pipeline to identify new insights into the microbial assemblage. Finally, a taxon‐based metabolic profile was used to predict the potential metabolic contribution of the prokaryotic community. Results indicated a refinement in terms of community composition using the ASV‐based bioinformatic methods. Functional profiling through 16S rRNA gene‐based analysis suggested metabolisms associated with carbonate precipitation, indicating a broad potential for microbially mediated carbonate precipitation (e.g., carbon fixation and photosynthesis). The possibility of biogenic methane gas production by methanogenic microorganisms was recognised, supporting the estimated flow of methane on the surface soils. The lacustrine evolution over the last years and the extreme physicochemical characteristics of RP have an impact on the microbial community structure. Prokaryotic community and metabolic potential results from RP coincide with the diversity and abundance of microbial communities and functional metabolisms reported from other microbialite‐forming lakes along the Trans Mexican Volcanic Belt. The identification of important possible phylotypes involved in carbonate precipitation might be an important factor in considering RP microbialites as active calcifying entities. These findings provide novel insights into the potential key role of metabolic pathways driving the process of carbonate precipitation inside RP and evidence of its importance as a microbial biodiversity hotspot in Mexico.

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“…The second approach, that is, de novo or distance‐based clustering (Navas‐Molina et al, 2013; Schloss & Westcott, 2011), clusters the sequences into OTUs based on the distance between the sequences themselves, instead of using the distance to a particular sequence in a database, and is often useful in the case of novel and unexplored habitats that may harbor untapped microbial diversity. De novo clustering methods suffer from the subjective nature of similarity threshold values for clustering and require increased computational power to process but may be able to better deal with limited sequence overlaps and ambiguous bases (Jeske & Gallert, 2022). Unfortunately, as OTUs generated from de novo methods are only compared to the sequences within the single data set, the OTUs are not able to be compared to external data, limiting the reproducibility of OTUs across different data sets (Callahan et al, 2017).…”

Section: Data Processingmentioning

confidence: 99%

“…The choice of pipeline, that is, clustering or denoising based, can have a significant impact on any resultant downstream analysis (Chiarello et al, 2022). However, Jeske and Gallert (2022) assessed the same data set with both OTU (VSEARCH—de novo clustering)—and ASV (DADA2)‐based approaches, and they found both methods to produce comparable outcomes that allowed for the same general interpretations to be made. They note that the resultant community compositions differed between 6.75% and 10.81% between pipelines, showcasing that while broad conclusions may be similar, such differences could interfere with downstream analysis depending on the sensitivity of any selected endpoints.…”

Section: Data Processingmentioning

confidence: 99%

Considerations for the inclusion of metabarcoding data in the plant protection product risk assessment of the soil microbiome

Sweeney

Kaushik

Bottoms

2023

Integr Envir Assess & Manag

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There is increasing interest in further developing the plant protection product (PPP) environmental risk assessment, particularly within the European Union, to include the assessment of soil microbial community composition, as measured by metabarcoding approaches. However, to date, there has been little discussion as to how this could be implemented in a standardized, reliable, and robust manner suitable for regulatory decision‐making. Introduction of metabarcoding‐based assessments of the soil microbiome into the PPP risk assessment would represent a significant increase in the degree of complexity of the data that needs to be processed and analyzed in comparison to the existing risk assessment on in‐soil organisms. The bioinformatics procedures to process DNA sequences into community compositional data sets currently lack standardization, while little information exists on how these data should be used to generate regulatory endpoints and the ways in which these endpoints should be interpreted. Through a thorough and critical review, we explore these challenges. We conclude that currently, we do not have a sufficient degree of standardization or understanding of the required bioinformatics and data analysis procedures to consider their use in an environmental risk assessment context. However, we highlight critical knowledge gaps and the further research required to understand whether metabarcoding‐based assessments of the soil microbiome can be utilized in a statistically and ecologically relevant manner within a PPP risk assessment. Only once these challenges are addressed can we consider if and how we should use metabarcoding as a tool for regulatory decision‐making to assess and monitor ecotoxicological effects on soil microorganisms within an environmental risk assessment of PPPs. Integr Environ Assess Manag 2023;00:1–22. © 2023 SETAC

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Microbiome Analysis via OTU and ASV-Based Pipelines—A Comparative Interpretation of Ecological Data in WWTP Systems

Cited by 26 publications

References 54 publications

Environmental DNA and RNA in aquatic community ecology: Toward methodological standardization

Environmental DNA and RNA in aquatic community ecology: Toward methodological standardization

Geomicrobiology of the Rincon de Parangueo maar crater: Exploring the link between an evolving extreme environment and its potential metabolic diversity

Considerations for the inclusion of metabarcoding data in the plant protection product risk assessment of the soil microbiome

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