Microfluidic qPCR Enables High Throughput Quantification of Microbial Functional Genes but Requires Strict Curation of Primers

Crane, Sally; Dorst, Josie van; Hose, Grant C.; King, Catherine K.; Ferrari, Belinda C.

doi:10.3389/fenvs.2018.00145

Cited by 27 publications

(17 citation statements)

References 89 publications

(76 reference statements)

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“…Most of the surveys of N-cycling functional markers in Antarctic soils (summarised in Figure 1 ) have focused on the core genes involved in N-fixation ( nifH ), nitrification ( amoA ) and denitrification processes ( narG for nitrate reduction, nirK and nirS for nitrite reduction, norB for nitric oxide reduction and nosZ for nitrous oxide reduction). Shotgun metagenomic and amplicon sequencing approaches have been used to explore the presence/absence and diversity of key nitrogen cycling genes [ 72 , 73 ], while gene abundances have been monitored using qPCR and its variations [ 25 , 42 , 74 , 75 , 76 ] and Geochip microarray technologies [ 34 , 68 , 77 ].…”

Section: N-cycling Genes In Soilsmentioning

confidence: 99%

Microbial Nitrogen Cycling in Antarctic Soils

et al. 2020

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The Antarctic continent is widely considered to be one of the most hostile biological habitats on Earth. Despite extreme environmental conditions, the ice-free areas of the continent, which constitute some 0.44% of the total continental land area, harbour substantial and diverse communities of macro-organisms and especially microorganisms, particularly in the more “hospitable” maritime regions. In the more extreme non-maritime regions, exemplified by the McMurdo Dry Valleys of South Victoria Land, nutrient cycling and ecosystem servicing processes in soils are largely driven by microbial communities. Nitrogen turnover is a cornerstone of ecosystem servicing. In Antarctic continental soils, specifically those lacking macrophytes, cold-active free-living diazotrophic microorganisms, particularly Cyanobacteria, are keystone taxa. The diazotrophs are complemented by heterotrophic bacterial and archaeal taxa which show the genetic capacity to perform elements of the entire N cycle, including nitrification processes such as the anammox reaction. Here, we review the current literature on nitrogen cycling genes, taxa, processes and rates from studies of Antarctic soils. In particular, we highlight the current gaps in our knowledge of the scale and contribution of these processes in south polar soils as critical data to underpin viable predictions of how such processes may alter under the impacts of future climate change.

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Section: N-cycling Genes In Soilsmentioning

confidence: 99%

Microbial Nitrogen Cycling in Antarctic Soils

et al. 2020

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“…Absolute quantification can be performed by coupling metabarcoding community data with total abundance data (Dannemiller et al 2014;Vandeputte et al 2017;Props et al 2017), developing microfluidic quantitative chips targeting pathobiome members (Kleyer et al, 2017;Crane et al, 2018) and by using meta-genomic approaches (Karasov et al 2019). These approaches will improve the reliability of pathobiome networks because they help reduce compositionality bias, i.e.…”

Section: Discussionmentioning

confidence: 99%

Microbial association networks give relevant insights into plant pathobiomes

Pauvert

Fort

Calonnec

et al. 2020

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Interactions between plant pathogens and other plant-associated microorganisms regulate disease.Deciphering the networks formed by these interactions, termed pathobiomes, is crucial to disease management. Our aim was to investigate whether microbial association networks inferred from metabarcoding data give relevant insights into pathobiomes, by testing whether inferred associations contain signals of ecological interactions. We used Poisson Lognormal Models to construct microbial association networks from metabarcoding data and then investigated whether some of these associations corresponded to interactions measurable in co-cultures or known in the literature, by using grapevine (Vitis vinifera) and the fungal pathogen causing powdery mildew (Erysiphe necator) as a model system. Our model suggested that the pathogen species was associated with 23 other fungal species, forming its putative pathobiome. These associations were not known as interactions in the literature, but one of them was confirmed by our co-culture experiments. The yeast Buckleyzyma aurantiaca impeded pathogen growth and reproduction, in line with the negative association found in the microbial network. Co-cultures also supported another association involving two yeast species. Together, these findings indicate that microbial networks can provide plausible hypotheses of ecological interactions that could be used to develop microbiome-based strategies for crop protection. Pauvert et al. 2020. Pathobiome networks 3

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“…In comparison to metagenome sequencing, HT-qPCR approaches have the potential to be more affordable and sensitive due to the targeted amplification of genes of interest and can be used for standardized surveys of microbial communities and their functions (31). The opportunities of HT-qPCR approaches and ampliconbased approaches depends strongly on the reliability of primer design to target genes of interest (32). In this present work, we introduce the MetaFunPrimer pipeline for designing HT-qPCR primers and demonstrate its use by capturing a broad diversity of relevant genes associated with ammonia oxidation within soil metagenomes.…”

Section: Discussionmentioning

confidence: 99%

MetaFunPrimer: primer design for targeting genes observed in metagenomes

Liu

Villanueva

Choi

et al. 2020

Preprint

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ABSTRACTHigh throughput primer design is needed to simultaneously design primers for multiple genes of interest, such as a group of functional genes. We have developed MetaFunPrimer, a bioinformatic pipeline to design primer targets for genes of interests, with a prioritization based on ranking the presence of gene targets in references, such as metagenomes. MetaFunPrimer takes inputs of protein and nucleotide sequences for gene targets of interest accompanied by a set of reference metagenomes or genomes for determining genes of interest. Its output is a set of primers that may be used to amplify genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 HT-qPCR primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 soil metagenomes. We demonstrate that these primers can significantly improve targeting of amoA-AOB genes in soil metagenomes compared to previously published primers.IMPORTANCEAmplification-based gene characterization allows for sensitive and specific quantification of functional genes. Often, there is a large diversity of genes represented for a function of interest, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high throughput primer design for functional genes of interest and also allows for ranking gene targets by their presence and abundance in environmental datasets. This tool enables high throughput qPCR approaches for characterizing functional genes.

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Microfluidic qPCR Enables High Throughput Quantification of Microbial Functional Genes but Requires Strict Curation of Primers

Cited by 27 publications

References 89 publications

Microbial Nitrogen Cycling in Antarctic Soils

Microbial Nitrogen Cycling in Antarctic Soils

Microbial association networks give relevant insights into plant pathobiomes

MetaFunPrimer: primer design for targeting genes observed in metagenomes

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