Steps in Metagenomics: Let’s Avoid Garbage in and Garbage Out

Izard, Jacques

doi:10.1016/b978-0-12-410472-3.00001-4

“…Any microbiome study, also called phytobiome for plants, using HTS can include methodological biases at each step of the analysis, i.e., during: (i) microbiota harvesting, (ii) sample storage/preservation, (iii) sample preparation (DNA extraction and library preparation), (iv) sequencing, (v) bioinformatics analysis, and (vi) data repository and experiment documentation in databases [10][11][12]. In order to efficiently use the increasing resources devoted to phytobiome studies, it is therefore of prime importance to pay careful attention to these methodological biases [13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Standardization of Plant Microbiome Studies: Which Proportion of the Microbiota is Really Harvested?

Sare

¹

,

Stouvenakers

²

,

Eck

³

et al. 2020

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Studies in plant-microbiome currently use diverse protocols, making their comparison difficult and biased. Research in human microbiome have faced similar challenges, but the scientific community proposed various recommendations which could also be applied to phytobiome studies. Here, we addressed the isolation of plant microbiota through apple carposphere and lettuce root microbiome. We demonstrated that the fraction of the culturable epiphytic microbiota harvested by a single wash might only represent one-third of the residing microbiota harvested after four successive washes. In addition, we observed important variability between the efficiency of washing protocols (up to 1.6-fold difference for apple and 1.9 for lettuce). QIIME2 analysis of 16S rRNA gene, showed a significant difference of the alpha and beta diversity between protocols in both cases. The abundance of 76 taxa was significantly different between protocols used for apple. In both cases, differences between protocols disappeared when sequences of the four washes were pooled. Hence, pooling the four successive washes increased the alpha diversity for apple in comparison to a single wash. These results underline the interest of repeated washing to leverage abundance of microbial cells harvested from plant epiphytic microbiota whatever the washing protocols, thus minimizing bias.

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“…This expression profile in a community allows the dynamics of gene expression patterns over time or environmental parameters to be realised, improving our understanding of the structure and function and adaptive mechanisms in complex communities (Parro & Paz, 2014). Whole-transcriptome analysis with total RNA sequencing captures a broad range of gene expression changes in that moment of time and enables the detection of novel transcripts in both coding and non-coding RNA species (Izard, 2015).…”

Section: Molecular Approaches To Unravel a Complex Microbial Communitymentioning

confidence: 99%

Microbiota of an Invasive Wasp Vespula vulgaris and Hymenopteran relatives: Interpreting the microbiome

Quinn¹

0

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<p>Invasive species represent a critical threat to ecosystems and ecological communities, causing changes through overwhelming predation as well as competing with native species for resources. Understanding the mechanisms behind invasive success is essential for understanding why they invade and the consequences of their invasions. Furthermore, invasive species, like all macroscopic organisms, harbour symbiotic and pathogenic microbes that constitute their microbiomes, which could explain invasive success. The complex ecological interaction networks within the microbiome can have a positive or negative impact on host abundance and dominance. These interactions may be significant for invasive species, where microbial influences acting on an exotic host can potentially drive the ecological success of an invasive population to the detriment of recipient communities. This thesis explores the microbiota of one of the most globally invasive species, the common wasp Vespula vulgaris, with the overall aim to investigate and characterise the microbiome of V. vulgaris, using metagenomics, bioinformatics and molecular techniques. The initial comparative microbiota study focused on three distinct life stages (larvae, worker and queen), from two ranges. This analysis revealed a core bacteriome community present in V. vulgaris. There was evidence of higher microbial diversity in wasp larvae compared with workers and queens. The Queen (gyne) microbiome revealed a more specific microbiome with absences of certain microbiota found in larvae and workers from the same nest, indicating a more distinctive microbiome. Interestingly, analysis of life stages between ranges showed significant dissimilarity in microbiomes, with microbiota loses, and acquisitions in the introduced New Zealand range. Using the same techniques, the microbiota of V. vulgaris and four hymenopteran hosts (Apis mellifera, Bombus terrestris, Vespula germanica and Linepithema humile), were comparatively analysed. The analysis investigated both shared microbiota and host specific microbiota. This analysis indicated the polyphagous V. vulgaris as having a diverse microbiome varying between nests and sites, indicating less specific microbiota in comparison to other hymenopteran hosts in this study. Vespid wasp colonies are known to occasionally crash in the presence of diseases; however, there is a lack of reliable evidence indicating pathogenic micro-organisms play an essential role in wasp colony dynamics. Incorporating knowledge gained in previous analyses, the next aim was to investigate V. vulgaris nests symptomatic of an infectious agent to discover the cause of pathology. Through molecular techniques, such as Illumina RNA-Seq, PCR and Sanger sequencing, the potential cause of infection and decline of diseased nests was examined. The metatranscriptomic comparison of diseased and healthy larvae highlighted five putative infectious agents. The bacteria Moellerella wisconsensis, Moku virus, Kashmir Bee Virus, Aspergillus and the microsporidian Vavraia culicis floridensis found in infected larvae, potentially causing pathology in the host. The first known instance of Moku virus, and potentially V. culicis floridensis and M. wisconsensis was documented as potential pathogens of V. vulgaris present in New Zealand. To test for potential virulence of these putative infectious agents, an infection study was carried out. Vespula vulgaris nests and larvae were orally infected in the lab using homogenised infected larvae. Subsequently, test and control larvae were sampled to conduct and quantify a time series analysis of infection using RT-qPCR using designed primers. This dissertation provided the first insight into the microbiome of V. vulgaris in the native and introduced range providing a baseline for further research. This analysis and the subsequent microbiota identified may play a role in wasp population dynamics, giving a better understanding of the observed thriving V. vulgaris population dynamics in New Zealand.</p>

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“…This expression profile in a community allows the dynamics of gene expression patterns over time or environmental parameters to be realised, improving our understanding of the structure and function and adaptive mechanisms in complex communities (Parro & Paz, 2014). Whole-transcriptome analysis with total RNA sequencing captures a broad range of gene expression changes in that moment of time and enables the detection of novel transcripts in both coding and non-coding RNA species (Izard, 2015).…”

Section: Molecular Approaches To Unravel a Complex Microbial Communitymentioning

confidence: 99%

Microbiota of an Invasive Wasp Vespula vulgaris and Hymenopteran relatives: Interpreting the microbiome

Quinn¹

0

View full text Add to dashboard Cite

<p>Invasive species represent a critical threat to ecosystems and ecological communities, causing changes through overwhelming predation as well as competing with native species for resources. Understanding the mechanisms behind invasive success is essential for understanding why they invade and the consequences of their invasions. Furthermore, invasive species, like all macroscopic organisms, harbour symbiotic and pathogenic microbes that constitute their microbiomes, which could explain invasive success. The complex ecological interaction networks within the microbiome can have a positive or negative impact on host abundance and dominance. These interactions may be significant for invasive species, where microbial influences acting on an exotic host can potentially drive the ecological success of an invasive population to the detriment of recipient communities. This thesis explores the microbiota of one of the most globally invasive species, the common wasp Vespula vulgaris, with the overall aim to investigate and characterise the microbiome of V. vulgaris, using metagenomics, bioinformatics and molecular techniques. The initial comparative microbiota study focused on three distinct life stages (larvae, worker and queen), from two ranges. This analysis revealed a core bacteriome community present in V. vulgaris. There was evidence of higher microbial diversity in wasp larvae compared with workers and queens. The Queen (gyne) microbiome revealed a more specific microbiome with absences of certain microbiota found in larvae and workers from the same nest, indicating a more distinctive microbiome. Interestingly, analysis of life stages between ranges showed significant dissimilarity in microbiomes, with microbiota loses, and acquisitions in the introduced New Zealand range. Using the same techniques, the microbiota of V. vulgaris and four hymenopteran hosts (Apis mellifera, Bombus terrestris, Vespula germanica and Linepithema humile), were comparatively analysed. The analysis investigated both shared microbiota and host specific microbiota. This analysis indicated the polyphagous V. vulgaris as having a diverse microbiome varying between nests and sites, indicating less specific microbiota in comparison to other hymenopteran hosts in this study. Vespid wasp colonies are known to occasionally crash in the presence of diseases; however, there is a lack of reliable evidence indicating pathogenic micro-organisms play an essential role in wasp colony dynamics. Incorporating knowledge gained in previous analyses, the next aim was to investigate V. vulgaris nests symptomatic of an infectious agent to discover the cause of pathology. Through molecular techniques, such as Illumina RNA-Seq, PCR and Sanger sequencing, the potential cause of infection and decline of diseased nests was examined. The metatranscriptomic comparison of diseased and healthy larvae highlighted five putative infectious agents. The bacteria Moellerella wisconsensis, Moku virus, Kashmir Bee Virus, Aspergillus and the microsporidian Vavraia culicis floridensis found in infected larvae, potentially causing pathology in the host. The first known instance of Moku virus, and potentially V. culicis floridensis and M. wisconsensis was documented as potential pathogens of V. vulgaris present in New Zealand. To test for potential virulence of these putative infectious agents, an infection study was carried out. Vespula vulgaris nests and larvae were orally infected in the lab using homogenised infected larvae. Subsequently, test and control larvae were sampled to conduct and quantify a time series analysis of infection using RT-qPCR using designed primers. This dissertation provided the first insight into the microbiome of V. vulgaris in the native and introduced range providing a baseline for further research. This analysis and the subsequent microbiota identified may play a role in wasp population dynamics, giving a better understanding of the observed thriving V. vulgaris population dynamics in New Zealand.</p>

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Steps in Metagenomics: Let’s Avoid Garbage in and Garbage Out

Cited by 5 publications

References 71 publications

Standardization of Plant Microbiome Studies: Which Proportion of the Microbiota is Really Harvested?

Standardization of Plant Microbiome Studies: Which Proportion of the Microbiota is Really Harvested?

Microbiota of an Invasive Wasp Vespula vulgaris and Hymenopteran relatives: Interpreting the microbiome

Microbiota of an Invasive Wasp Vespula vulgaris and Hymenopteran relatives: Interpreting the microbiome

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