Optimization of 16S amplicon analysis using mock communities: implications for estimating community diversity

Krohn, Andrew; Stevens, Bo; Robbins‐Pianka, Adam; Belus, Matthew; Allan, Gerard J.; Gehring, Catherine A.

doi:10.7287/peerj.preprints.2196v2

Cited by 4 publications

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“…By contrast to the single-copy housekeeping gene targeted by the rpoB marker, sequence heterogeneity between the different copies of the16S marker may lead to the amplification of numerous sequence variants during metabarcoding, resulting in the identification of excessive numbers of OTUs in 16S datasets. Intragenomic ribosomal diversity [25] or ribosomal paralogs [26] have frequently been implicated as the major source of sequence variants in 16S Illumina A B Fig. 3 Comparison of the expected bacterial composition and the observed OTU composition obtained with Illumina-amplicon rpoB sequencing in the mock1 community (FROGS process).…”

Section: Discussionmentioning

confidence: 99%

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

OGIER

Pagès

Galan

et al. 2019

Preprint

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Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

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Section: Discussionmentioning

confidence: 99%

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

OGIER

Pagès

Galan

et al. 2019

Preprint

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“…n = 1194) of the total read abundance on a per-sample basis and that were present in < 5% (i.e. n = 12) of the total samples (Bokulich et al, 2013;Callahan et al, 2016;Krohn et al, 2016). Only OTUs assigned to the algal genus Trebouxia passed the filtering process and were thus retained for downstream analysis.…”

Section: Bioinformatic Analysesmentioning

confidence: 99%

“…The main drawbacks are those related to primer biases, sequencing artifacts, clustering of operational taxonomic units, and contamination (Thomsen & Willerslev, 2015;Guardiola et al, 2016). Thus, care should be taken during sample collection and preparation, followed by the application of strict bioinformatic filtering steps (Krohn et al, 2016). This is particularly relevant in the case of lichens and their photobionts, to avoid or minimize the inclusion of epiphytic algae that may have landed on or within the thallus during the life cycle of the lichen but that are not a functional part of the symbiosis.…”

Section: Introductionmentioning

confidence: 99%

Environment and host identity structure communities of green algal symbionts in lichens

et al. 2017

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SummaryAn understanding of how biotic interactions shape species' distributions is central to predicting host-symbiont responses under climate change. Switches to locally adapted algae have been proposed to be an adaptive strategy of lichen-forming fungi to cope with environmental change. However, it is unclear how lichen photobionts respond to environmental gradients, and whether they play a role in determining the fungal host's upper and lower elevational limits.Deep-coverage Illumina DNA metabarcoding was used to track changes in the community composition of Trebouxia algae associated with two phylogenetically closely related, but ecologically divergent fungal hosts along a steep altitudinal gradient in the Mediterranean region.We detected the presence of multiple Trebouxia species in the majority of thalli. Both altitude and host genetic identity were strong predictors of photobiont community assembly in these two species. The predominantly clonally dispersing fungus showed stronger altitudinal structuring of photobiont communities than the sexually reproducing host. Elevation ranges of the host were not limited by the lack of compatible photobionts.Our study sheds light on the processes guiding the formation and distribution of specific fungal-algal combinations in the lichen symbiosis. The effect of environmental filtering acting on both symbiotic partners appears to shape the distribution of lichens.

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“…By contrast to the single-copy housekeeping gene targeted by the rpoB marker, sequence heterogeneity between the different copies of the16S marker may lead to the amplification of numerous sequence variants during metabarcoding, resulting in the identification of excessive numbers of OTUs in 16S datasets. Intragenomic ribosomal diversity [25] or ribosomal paralogs [26] have frequently been implicated as the major source of sequence variants in 16S Illumina amplicon-sequencing analyses. The observed OTU inflation may also be explained by cumulative errors occurring during the amplification and sequencing steps of the metabarcoding procedure, resulting in the detection of sequence variants.…”

Section: Discussionmentioning

confidence: 99%

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

Ogier

Pagès

Galan

et al. 2019

Preprint

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Optimization of 16S amplicon analysis using mock communities: implications for estimating community diversity

Abstract: 33The diversity of complex microbial communities can be rapidly assessed by high- defining OTUs depended on the method used for OTU picking. Our "default" analysis in QIIME 45 overestimated mock community OTU diversity by at least a factor of ten. Our optimized analysis

Cited by 4 publications

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rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

Environment and host identity structure communities of green algal symbionts in lichens

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

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