Misguided phylogenetic comparisons using DGGE excised bands may contaminate public sequence databases

Pylro, Victor Satler; Morais, Daniel; Kalks, Karlos Henrique Martins; Roesch, Luiz Fernando Würdig; Hirsch, P. R.; Tótola, Marcos Rogério; Yotoko, Karla Suemy Clemente

doi:10.1016/j.mimet.2016.04.012

Cited by 3 publications

(5 citation statements)

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“…Even though the sequencing analysis of microbial communities by 16S rRNA gene display more accurate results than fingerprinting methods (for example, DGGE band sequencing) (Pylro et al, 2016) this analysis also has some limitations due that the identity of microorganisms depends on reported sequences within databases. Because of that, it is recomendable to complement this information by studying functional genes (Oka et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Correlations between microbial population dynamics, bamA gene abundance and performance of anaerobic sequencing batch reactor (ASBR) treating increasing concentrations of phenol

Franchi

Cabrol

Chamy

et al. 2020

Journal of Biotechnology

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The relevant microorganims driving efficiency changes in anaerobic digestion of phenol remains uncertain. In this study correlations were established between microbial population and the process performance in an anaerobic sequencing batch reactor (ASBR) treating increasing concentrations of phenol (from 120 to 1200 mg L −1 ). Sludge samples were taken at different operational stages and microbial community dynamics was analyzed by 16S rRNA sequencing. In addition, bamA gene was quantified in order to evaluate the dynamics of anaerobic aromatic degraders. The microbial community was dominated by Anaerolineae, Bacteroidia, Clostridia, and Methanobacteria classes. Correlation analysis between bamA gene copy number and phenol concentration were highly significant, suggesting that the increase of aromatic degraders targeted by bamA assay was due to an increase in the amount of phenol degraded over time. The incremental phenol concentration affected hydrogenotrophic archaea triggering a linear decrease of Methanobacterium and the growth of Methanobrevibacter. The best performance in the reactor was at 800 mg L −1 of phenol. At this stage, the highest relative abundances of Syntrophorhabdus, Chloroflexus, Smithella, Methanolinea and Methanosaeta were observed and correlated positively with initial degradation rate, suggesting that these microorganisms are relevant players to maintain a good performance in the ASBR.

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Section: Introductionmentioning

confidence: 99%

Correlations between microbial population dynamics, bamA gene abundance and performance of anaerobic sequencing batch reactor (ASBR) treating increasing concentrations of phenol

Franchi

Cabrol

Chamy

et al. 2020

Journal of Biotechnology

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“…The strategy we used, however, harbours issues with varying gene copy numbers within the same genus that forces GCN to use averages. In addition to that, multiple copies in the same gene can diverge [15] as a result of pseudogene formation or horizontal gene transfer [16]. One of the eleven mock communities (Mock-12) showed a poor fit of the sequencing data to the mock community (shown in red in Table 1) and was therefore removed from the analysis (Fig.…”

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confidence: 99%

16S rRNA Gene Copy Number Normalization Does Not Provide More Reliable Conclusions in Metataxonomic Surveys

2020

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Sequencing 16S rRNA gene amplicons is the gold standard to uncover the composition of prokaryotic communities. The presence of multiple copies of this gene makes the community abundance data distorted and gene copy normalization (GCN) necessary for correction. Even though GCN of 16S data provided a picture closer to the metagenome before, it should also be compared with communities of known composition due to the fact that library preparation is prone to methodological biases. Here, we process 16S rRNA gene amplicon data from eleven simple mock communities with DADA2 and estimate the impact of GCN. In all cases, the mock community composition derived from the 16S sequencing differs from those expected, and GCN fails to improve the classification for most of the analysed communities. Our approach provides empirical evidence that GCN does not improve the 16S target sequencing analyses in real scenarios. We therefore question the use of GCN for metataxonomic surveys until a more comprehensive catalogue of copy numbers becomes available. Keywords 16S rRNA. Metataxonomic surveys. Gene Amplicon sequencing of 16S rRNA gene is considered a gold standard to evaluate the composition of prokaryotic communities due to (i) low cost, (ii) easy availability, (iii) easy practicality of extraction and preparation kits, (iv) high taxonomic resolution as deep as the level of genera (or sometimes species) and (v) extensive databases. The concept of gold standards implies a level of perfection never attained by any biological test [1] which is why those are constantly challenged and replaced when appropriate [2]. Still, amplicon sequencing outcompetes (88,889 papers with "16S rRNA" as of

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“…Additionally, ITS2-DGGE has shown low taxonomic resolution to distinguish many recently identified hostspecific lineages or IGVs identified by higher-resolution markers (e.g., [4,19,73]). Recent integrative approaches that rely upon multiple lines of evidence are supporting robust species hypotheses and clarifying species delineations in Symbiodiniaceae [18][19][20][21]57].…”

Section: Limitations and General Considerationsmentioning

confidence: 99%

“…Additionally, about 15% of the association records were identified using cloning of rDNA genes (Table S1), which is known to increase the variability of the sequences generated and artificially increase richness estimates [101]; nevertheless, because many of the phylotypes identified by cloning were common to the main phylotypes identified by ITS2-DGGE and artifacts are typically rare events, we expect them to have a negligible influence on the conclusions of this study. Furthermore, while most DGGE bands were excised, sequenced, and reported as containing unique phylotypes, or particular configurations of phylotypes if co-dominant bands were present (Table S1), IGVs may artificially overestimate richness in particular assemblages or may not be resolved by DGGE and underestimate richness [73]. However, in spite of these drawbacks, ITS2-DGGE has identified many phylotypes over the last decades that have proven congruent with higherresolution genetic markers (e.g., [18]) and several compilations of coral-Symbiodiniaceae associations (e.g., [5,102,103], which are included in our dataset) have been analyzed to inform general trends and patterns.…”

Section: Limitations and General Considerationsmentioning

confidence: 99%

“…We then perform comparative analysis of their correlations with bleaching susceptibility across 123 coral species using phylogenetic comparative methods. We chose the ITS2-DGGE phylotypes for our analyses because it is the most extensive dataset currently available, with the understanding that (i) we are conservatively estimating richness as we are limited to the most abundant (marker sensitivity of1 0%) and likely most physiologically relevant phylotypes, and (ii) we will not have the best available resolving power to distinguish specific lineages or intragenomic variants identified by higher-resolution markers (e.g., [4,19,73]). We compiled and analyzed a dataset of 15,566 records of associations between 123 coral species with documented responses to thermal stress [74] and 377 Symbiodiniaceae ITS2 phylotypes.…”

Section: Introductionmentioning

confidence: 99%

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Uncovering the role of Symbiodiniaceae assemblage composition and abundance in coral bleaching response by minimizing sampling and evolutionary biases

et al. 2020

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Background Biodiversity and productivity of coral-reef ecosystems depend upon reef-building corals and their associations with endosymbiotic Symbiodiniaceae, which offer diverse functional capabilities to their hosts. The number of unique symbiotic partners (richness) and relative abundances (evenness) have been hypothesized to affect host response to climate change induced thermal stress. Symbiodiniaceae assemblages with many unique phylotypes may provide greater physiological flexibility or form less stable symbioses; assemblages with low abundance phylotypes may allow corals to retain thermotolerant symbionts or represent associations with less-suitable symbionts. Results Here we demonstrate that true richness of Symbiodiniaceae phylotype assemblages is generally not discoverable from direct enumeration of unique phylotypes in association records and that cross host-species comparisons are biased by sampling and evolutionary patterns among species. These biases can be minimized through rarefaction of richness (rarefied-richness) and evenness (Probability of Interspecific Encounter, PIE), and analyses that account for phylogenetic patterns. These standardized metrics were calculated for individual Symbiodiniaceae assemblages composed of 377 unique ITS2 phylotypes associated with 123 coral species. Rarefied-richness minimized correlations with sampling effort, while maintaining important underlying characteristics across host bathymetry and geography. Phylogenetic comparative methods reveal significant increases in coral bleaching and mortality associated with increasing Symbiodiniaceae assemblage richness and evenness at the level of host species. Conclusions These results indicate that the potential flexibility afforded by assemblages characterized by many phylotypes present at similar relative abundances does not result in decreased bleaching risk and point to the need to characterize the overall functional and genetic diversity of Symbiodiniaceae assemblages to quantify their effect on host fitness under climate change.

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Misguided phylogenetic comparisons using DGGE excised bands may contaminate public sequence databases

Cited by 3 publications

References 34 publications

Correlations between microbial population dynamics, bamA gene abundance and performance of anaerobic sequencing batch reactor (ASBR) treating increasing concentrations of phenol

Correlations between microbial population dynamics, bamA gene abundance and performance of anaerobic sequencing batch reactor (ASBR) treating increasing concentrations of phenol

16S rRNA Gene Copy Number Normalization Does Not Provide More Reliable Conclusions in Metataxonomic Surveys

Uncovering the role of Symbiodiniaceae assemblage composition and abundance in coral bleaching response by minimizing sampling and evolutionary biases

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