Metagenomics vs. total RNA sequencing: most accurate data-processing tools, microbial identification accuracy, and implications for freshwater assessments

Hempel, Christopher; Wright, Natalie Andrews; Harvie, Julia; Hleap, Jose Sergio; Adamowicz, Sarah J.; Steinke, Dirk

doi:10.1101/2022.06.03.494701

Cited by 5 publications

(7 citation statements)

References 108 publications

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“…The similarity in community patterns also supports our previous conclusion that the impact of primer bias in amplicon approaches is negligible at higher taxonomic levels of analysis. Shotgun approaches are, therefore, highly promising and could be further functionalized by stepping toward an RNA-focused metatranscriptomic approach [ 62 , 63 ].…”

Section: Discussionmentioning

confidence: 99%

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Rieder

Kapopoulou

Bank

et al. 2023

Environmental Microbiome

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Background Microbial communities in recirculating aquaculture systems (RAS) play a role in system success, nutrient cycling, and water quality. Considering the increasing socio-economic role of fish farming, e.g., regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective, especially regarding the growth, development, and welfare of the farmed animal. However, the current data on the composition of microbial communities within RAS is patchy, which is partly attributable to diverging method choices that render comparative analyses challenging. Therefore, there is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems. Results We compared sequencing approach performances (3 types of 16S short amplicon sequencing, PacBio long-read amplicon sequencing, and amplification-free shotgun metagenomics) in the characterization of microbial communities in two commercial RAS fish farms. Results showed that 16S primer choice and amplicon length affect some values (e.g., diversity measures, number of assigned taxa or distinguishing ASVs) but have no impact on spatio-temporal patterns between sample types, farms and time points. This implies that 16S rRNA approaches are adequate for community studies. The long-read amplicons underperformed regarding the quantitative resolution of spatio-temporal patterns but were suited to identify functional services, e.g., nitrification cycling and the detection of pathogens. Finally, shotgun metagenomics extended the picture to fungi, viruses, and bacteriophages, opening avenues for exploring inter-domain interactions. All sequencing datasets agreed on major prokaryotic players, such as Actinobacteriota, Bacteroidota, Nitrospirota, and Proteobacteria. Conclusion The different sequencing approaches yielded overlapping and highly complementary results, with each contributing unique data not obtainable with the other approaches. We conclude that a tiered approach constitutes a strategy for obtaining the maximum amount of information on aquaculture microbial communities and can inform basic research on community evolution dynamics. For specific and/or applied questions, single-method approaches are more practical and cost-effective and could lead to better farm management practices.

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Section: Discussionmentioning

confidence: 99%

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Rieder

Kapopoulou

Bank

et al. 2023

Environmental Microbiome

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“…The latter further supports the above notion that the impact of primer bias in amplicon approaches is negligible at higher taxonomic levels. Shotgun approaches are, therefore, highly promising and could be further improved by stepping toward an RNA-focused metatranscriptomic approach [59], [60].…”

Section: Discussionmentioning

confidence: 99%

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Rieder

Kapopoulou

Bank

et al. 2022

Preprint

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Recirculating aquaculture systems (RAS) heavily depend on microbial communities to maintain water quality. These communities therefore influence the growth, development, and welfare of farmed fish. With the increasing socio-economic role of fish farming e.g. regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective. However, the data situation regarding the composition of microbial communities within RAS is patchy. Since this is partly ascribed to method choices, there clearly is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems.Here, we compare the performance of 16S amplicon sequencing, Pac-Bio long-read amplicon sequencing, and amplification-free shotgun metagenomics in the characterization of microbial communities in two commercial-size RAS fish farms. We show that, even though primer choice affects read quality, diversity, and assigned taxa, distinct primer pairs uncover similar spatio-temporal patterns between sample types, farms, and time points. We find that long-read amplicons underperform regarding quantitative resolution of spatio-temporal patterns, but allow for species-level identification of functional services and pathogens. Finally, shotgun metagenomics data identified fungi, viruses, and bacteriophages, opening avenues for an exploration of natural approaches regarding antipathogenic treatments. Overall, the datasets agreed on major prokaryotic players.In conclusion, different sequencing approaches yield overlapping and highly complementary results, with each contributing data no other approach could. Such a tiered approach therefore constitutes a practical and cost-effective strategy for obtaining the maximum amount of information on aquaculture microbial communities. These data could lead to better farm management practices and at the same time inform basic research on community evolution dynamics.

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“…The data used in this study originate from an earlier study, in which we applied total RNA-Seq and metagenomics and investigated 672 combinations of data-processing tools to identify the best-performing sequencing method and combination of tools to characterize taxonomically a mock microbial community (Hempel et al, 2022). Details of the laboratory and bioinformatics processing steps can be found there (Hempel et al, 2022) Random subsampling was performed ten times, and the subsamples were processed independently.…”

Section: Laboratory and Bioinformatics Processingmentioning

confidence: 99%

“…In an earlier study, we were able to show that total RNA-Seq characterized a mock microbial community consisting of 10 species more accurately than metagenomics at almost one order of magnitude lower sequencing depth (Hempel et al, 2022). For the present study, we use the same data to test the performance of metagenomics and total RNA-Seq in terms of SSU rRNA completeness, i.e., the portion of the SSU rRNA that can be successfully reconstructed.…”

Section: Introductionmentioning

confidence: 99%

“…A few studies compared the performance of total RNA-Seq and metabarcoding (Lanzén et al, 2011;Yan et al, 2018) or metagenomics (Hempel et al, 2022;Lanzén et al, 2011;Shi, Tyson, Eppley, & Delong, 2011;Urich et al, 2014;Uyaguari-Diaz et al, 2016) for the analysis of microbial community composition. However, the use of total RNA-Seq to analyze microbial communities remains rare, and a comparison of total RNA-Seq and metagenomics in terms of SSU rRNA reconstruction is lacking, although the results of such a comparison could impact our ability to categorize global biodiversity and analyze microbial communities.…”

Section: Introductionmentioning

confidence: 99%

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Reconstruction of Small Subunit Ribosomal RNA from High-Throughput Sequencing Data: A Comparative Study of Metagenomics and Total RNA Sequencing

Hempel

Carson

Elliott

et al. 2022

Preprint

Self Cite

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The small subunit (SSU) ribosomal RNA (rRNA) is the most commonly used marker for the identification of microbial taxa, but its full-length reconstruction from high-throughput sequencing (HTS) data remains challenging, especially for complex and diverse environmental samples. Metagenomics and total RNA sequencing (total RNA-Seq) are target-PCR-free HTS methods that are used to characterize microbial communities and simultaneously reconstruct SSU rRNA sequences. However, more testing is required to determine and improve their effectiveness. In this study, we processed metagenomics and total RNA-Seq data retrieved from a commercially available mock microbial community using 112 combinations of commonly used data-processing tools, determined SSU rRNA reconstruction completeness of both sequencing methods for each species in the mock community, and analyzed the impact of data-processing tools on SSU rRNA and genome completeness. Total RNA-Seq allowed for the complete or near-complete reconstruction of all mock community SSU rRNA sequences and outperformed metagenomics. SSU rRNA completeness of metagenomics strongly correlated with the genome size of mock community species. The impact of data-processing tools was overall low, although certain tools resulted in significantly lower SSU rRNA completeness. These results are promising for the high-throughput reconstruction of novel full-length SSU rRNA sequences and could advance the simultaneous application of multiple -omics approaches in routine environmental assessments to allow for more holistic assessments of ecosystems.

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Metagenomics vs. total RNA sequencing: most accurate data-processing tools, microbial identification accuracy, and implications for freshwater assessments

Cited by 5 publications

References 108 publications

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

Reconstruction of Small Subunit Ribosomal RNA from High-Throughput Sequencing Data: A Comparative Study of Metagenomics and Total RNA Sequencing

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