Total RNA-sequencing reveals multi-level microbial community changes and functional responses to wood ash application in agricultural and forest soil

Bang‐Andreasen, Toke; Anwar, Muhammad Zohaib; Lanzén, Anders; Kjøller, Rasmus; Rønn, Regin; Jacobsen, Carsten Suhr

doi:10.1101/621557

Cited by 4 publications

(9 citation statements)

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“…In the second study, we investigated the effects of wood ash amendment on Danish forest soils [15]. Ash was added in 3 different quantities (0/control, 3, 12, and 90 tonnes ash per hectare [t ha −1 ]) and the effect over time was analysed in soil communities at 0, 3, 30, and 100 days after ash addition.…”

Section: Resultsmentioning

confidence: 99%

“…Three different protein databases were selected for this benchmarking in order to include a representative selection of 3 different degrees of specialization, on a range from a more inclusive database with wide coverage (universality) and low degree of expert curation to a smaller, highly curated database, with more narrow coverage: (i) M5nr [11], an inclusive and comprehensive non-redundant protein database in combination with Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOG) hierarchical annotation; (ii) Carbohydrate-Active Enzymes (CAZy) [12], a database dedicated to describing the families of structurally related catalytic and carbohydrate-binding modules of enzymes; and (iii) Nitrogen Cycling Database (NCycDB) [13], a specialized and manually curated database covering only nitrogen cycle genes. Finally, to estimate the consistency and variance in the results caused by the choice of approach, we then applied them to real-world metatranscriptomes from microbial communities in (i) active-layer permafrost soil from Svalbard, Norway [14], and (ii) ash-impacted Danish forest soil [15].…”

Section: Introductionmentioning

confidence: 99%

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To assemble or not to resemble—A validated Comparative Metatranscriptomics Workflow (CoMW)

et al. 2019

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Background Metatranscriptomics has been used widely for investigation and quantification of microbial communities’ activity in response to external stimuli. By assessing the genes expressed, metatranscriptomics provides an understanding of the interactions between different major functional guilds and the environment. Here, we present a de novo assembly-based Comparative Metatranscriptomics Workflow (CoMW) implemented in a modular, reproducible structure. Metatranscriptomics typically uses short sequence reads, which can either be directly aligned to external reference databases (“assembly-free approach”) or first assembled into contigs before alignment (“assembly-based approach”). We also compare CoMW (assembly-based implementation) with an assembly-free alternative workflow, using simulated and real-world metatranscriptomes from Arctic and temperate terrestrial environments. We evaluate their accuracy in precision and recall using generic and specialized hierarchical protein databases. Results CoMW provided significantly fewer false-positive results, resulting in more precise identification and quantification of functional genes in metatranscriptomes. Using the comprehensive database M5nr, the assembly-based approach identified genes with only 0.6% false-positive results at thresholds ranging from inclusive to stringent compared with the assembly-free approach, which yielded up to 15% false-positive results. Using specialized databases (carbohydrate-active enzyme and nitrogen cycle), the assembly-based approach identified and quantified genes with 3–5 times fewer false-positive results. We also evaluated the impact of both approaches on real-world datasets. Conclusions We present an open source de novo assembly-based CoMW. Our benchmarking findings support assembling short reads into contigs before alignment to a reference database because this provides higher precision and minimizes false-positive results.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

To assemble or not to resemble—A validated Comparative Metatranscriptomics Workflow (CoMW)

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the second study, we investigated the effects of wood ash amendment on Danish forest soils [15]. Ash was added in three different quantities (0/control, 3, 12 and 90 tonnes ash per hectare (t ha −1 )) and the effect over time was analysed in soil communities at 0, 3, 30 and 100 days after ash addition.…”

Section: Resultsmentioning

confidence: 99%

“…Though not shown here, we expect that the former approach would also benefit in accuracy from assembling mRNA to full length transcripts before classification, based on our results regarding functional diversity. The total RNA approach also benefits from custom rRNA targeted assembly [15], which may be incorporated into CoMW thanks to its modularity.…”

Section: Discussionmentioning

confidence: 99%

“…Three different protein databases were selected for this benchmarking in order to include a representative selection of three different degrees of specialization, on a range from a more inclusive database with wide coverage (universality) and low degree of expert curation, to a smaller, highly curated database, with more narrow coverage: 1) M5nr [11] an inclusive and comprehensive non-redundant protein database in combination with eggNOG hierarchical annotation 2) Carbohydrate-Active Enzymes (CAZymes) [12] a database dedicated to describing the families of structurally-related catalytic and carbohydrate-binding modules of enzymes and 3) Nitrogen Cycling Database (NCycDB) [13] a specialized and manually curated database covering only N cycle genes. Finally, in order to estimate the consistency and variance in the results caused by the choice of approach we then applied them to real world metatranscriptomes from microbial communities in 1) active-layer permafrost soil from Svalbard [14] and 2) Ash impacted Danish Forest soil [15].…”

Section: Introductionmentioning

confidence: 99%

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To assemble or not to resemble – A validated Comparative Metatranscriptomics Workflow (CoMW)

Anwar

Lanzén

Bang‐Andreasen

et al. 2019

Preprint

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BackgroundMetatranscriptomics has been used widely for investigation and quantification of microbial communities’ activity in response to external stimuli. By assessing the genes expressed, metatranscriptomics provide an understanding of the interactions between different major functional guilds and the environment. Here, we present de-novo assembly-based Comparative Metatranscriptomics Workflow (CoMW) implemented in a modular, reproducible structure, significantly improving the annotation and quantification of metatranscriptomes. Metatranscriptomics typically utilize short sequence reads, which can either be directly aligned to external reference databases (“assembly-free approach”) or first assembled into contigs before alignment (“assembly-based approach”). We also compare CoMW (assembly-based implementation) with assembly-free alternative workflow, using simulated and real-world metatranscriptomes from Arctic and Temperate terrestrial environments. We evaluate their accuracy in precision and recall using generic and specialized hierarchical protein databases.ResultsCoMW provided significantly fewer false positives resulting in more precise identification and quantification of functional genes in metatranscriptomes. Using the comprehensive database M5nr, the assembly-based approach identified genes with only 0.6% false positives at thresholds ranging from inclusive to stringent compared to the assembly-free approach yielding up to 15% false positives. Using specialized databases (Carbohydrate Active-enzyme and Nitrogen Cycle), the assembly-based approach identified and quantified genes with 3-5x less false positives. We also evaluated the impact of both approaches on real-world datasets.ConclusionsWe present an open source de-novo assembly-based Comparative Metatranscriptomics Workflow (CoMW). Our benchmarking findings support the argument of assembling short reads into contigs before alignment to a reference database, since this provides higher precision and minimizes false positives.

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Community RNA-Seq: Multi-kingdom responses to living versus decaying root inputs in soil

Nuccio

Nguyen

Rocha

et al. 2021

Preprint

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Roots are the primary source of organic carbon inputs to most soils. Decomposition is a multi-trophic process involving multiple kingdoms of microbial life, but typically microbial ecology studies focus on one or two major lineages in isolation. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis (“community RNA-Seq”) to simultaneously study the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter. We identified rhizosphere substrate preferences for 13C-exudates versus 15N-litter using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group’s more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving cross-kingdom interactions, and the trajectory of carbon through this soil food web likely impacts the fate of carbon in soil.

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Total RNA-sequencing reveals multi-level microbial community changes and functional responses to wood ash application in agricultural and forest soil

Abstract: 8Recycling of wood ash from energy production may counteract soil acidification and return 1 9

Cited by 4 publications

References 94 publications

To assemble or not to resemble—A validated Comparative Metatranscriptomics Workflow (CoMW)

To assemble or not to resemble—A validated Comparative Metatranscriptomics Workflow (CoMW)

To assemble or not to resemble – A validated Comparative Metatranscriptomics Workflow (CoMW)

Community RNA-Seq: Multi-kingdom responses to living versus decaying root inputs in soil

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