Accurate determination of microbial diversity from 454 pyrosequencing data

Quince, Christopher; Lanzén, Anders; Curtis, Tom; Davenport, Russell J.; Hall, Neil; Head, Ian M.; Read, L. F.; Sloan, William T.

doi:10.1038/nmeth.1361

Cited by 896 publications

(825 citation statements)

References 152 publications

(192 reference statements)

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“…We did not discard entire sequence reads containing low‐quality score sections, which would have resulted in too few remaining sequences; instead, those sequences were truncated to exclude the poor‐quality section. After initial quality check, we then employed QIIME's algorithms that approximate PyroNoise (Quince et al., 2009; Reeder & Knight, 2010) to cluster the flowgram (analogous to the trace data in Sanger sequencing) and denoise the data. Next, we removed forward primer sequences from the end of reads, allowing at most one mismatch to the primer sequence, again to minimize the number of discards, where reads with more than one mismatch to the forward primer sequences were removed.…”

Section: Methodsmentioning

confidence: 99%

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Thongsripong

Chandler

Green

et al. 2017

Ecology and Evolution

124

112

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Vector‐borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito‐associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next‐generation 454 pyrosequencing of 16S and 18S rRNA amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered (Ascogregarina and Wolbachia, respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human‐impacted landscapes that may ultimately affect vector‐borne disease risk.

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Section: Methodsmentioning

confidence: 99%

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Thongsripong

Chandler

Green

et al. 2017

Ecology and Evolution

124

112

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“…Processing of the resulting sequences, i.e. trimming and quality control, was performed with the MOTHUR software (v 1.30) (Schloss et al, 2009) including denoising of the flowgrams using PyroNoise (Quince et al 2009) and data normalization to the smallest number of sequences in the resulting libraries. Sequences ≥250 bp with no ambiguous base assignments and no homopolymers ≥8 bp were included in downstream analyses.…”

Section: Methodsmentioning

confidence: 99%

Inter-comparison of the potentially active prokaryotic communities in the halocline sediments of Mediterranean deep-sea hypersaline basins

et al. 2015

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The sediment microbiota of the Mediterranean deep--sea anoxic hypersaline basins (DHABs) are understudied relative to communities in the brines and halocline waters. In this study, the active fraction of the prokaryotic community in the halocline sediments of L' Atalante, Urania and Discovery DHABs was investigated based on extracted total RNA and 454 pyrosequencing of the 16S rRNA gene. Bacterial and archaeal communities were different in the sediments underlying the halocline waters of the three habitats, reflecting the unique chemical settings of each basin. The relative abundance of unique operational taxonomic units (OTUs) was also different between deep--sea control sediments and sediments underlying DHAB haloclines, suggesting adaptation to the steep DHAB chemical gradients. Only a few OTUs were affiliated to known bacterial halophilic and/or aerobic groups. Many OTUs, including some of the dominant ones, were related to aerobic taxa. Archaea were detected only in few halocline samples, with lower OTU richness relative to Bacteria, and were dominated by taxa associated with methane cycling. This study suggests that while metabolically active prokaryotic communities appear to be present in sediments underlying the three DHABs investigated, their diversity and activity are likely to be more reduced in sediments underlying the brines.3

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“…Other aspects, more on the technical side, are that primer selection may affect amplification of the community differently and the error attributed to the pyrosequencing technique itself that can lead to an overestimation of taxon abundance. If not corrected, this can be as much as 35% of the sequences (Gomez-Alvarez et al, 2009;Quince et al, 2009). Recently, the overestimation of taxa was illustrated when a single genome generated hundreds of different sequence types, leading to recommendations of strict quality filtering and careful application of sequence difference cut-offs for grouping sequences into operational taxa (Pukall et al, 2009;Purushe et al, 2010).…”

Section: Rumen Microbial Diversitymentioning

confidence: 99%

Rumen microbial (meta)genomics and its application to ruminant production

Morgavi

Kelly

Janssen

et al. 2013

Animal

209

163

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Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome–host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

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Accurate determination of microbial diversity from 454 pyrosequencing data

Cited by 896 publications

References 152 publications

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Inter-comparison of the potentially active prokaryotic communities in the halocline sediments of Mediterranean deep-sea hypersaline basins

Rumen microbial (meta)genomics and its application to ruminant production

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