Biology subjects

Cholet, F.

Publications and source records attributed to Cholet, F..

5 recordsLinked to original sources

bioRxiv2026

Activity-resolved microbial community profiling using rpoB gene and transcript sequencing

Determining which members of a microbial community are metabolically active remains a central challenge in microbial ecology. Although the 16S rRNA gene is the dominant marker for bacterial community profiling, it cannot reliably distinguish active cells from dormant or dead populations. As a result, complementary phylogenetic markers whose transcript abundance more closely reflects cellular activity are needed. Here, we systematically evaluated 80 Bacterial protein-coding marker genes and identified rpoB, encoding the beta subunit of bacterial RNA polymerase, as the optimal candidate. We designed a new primer pair (1528F 2041R) from a curated database of 305,274 unique rpoB sequences and validated it for quantitative PCR and amplicon sequencing of DNA and RNA templates. The rpoB qPCR assay achieved a limit of quantification two orders of magnitude lower than the benchmark 16S rRNA assay, for which a limit of detection could not be determined because of no-template-control amplification. In soil and sediment communities, rpoB recovered community composition comparable to 16S rRNA while providing a quantitative activity signal: rpoB cDNA:DNA ratios correlated significantly with taxon-level transcript abundance (R squared between 0.22 and 0.29, p < 0.001), whereas 16S rRNA ratios did not (p > 0.5). In a biological activated carbon biofilter experiment, rpoB transcript abundance tracked the decline in dissolved organic carbon removal rates across a 72 hour time series (correlation coefficients between 0.84 and 0.99), whereas 16S rRNA transcripts were uninformative (correlation coefficients between -0.4 and 0.98). These results establish rpoB as a quantitatively robust, activity-responsive complement to 16S rRNA for linking community composition to ecosystem processes.

microbiology↗

bioRxiv2023

intI1 primer selection for class 1 integron integrase gene and transcript quantification - validation and application for monitoring intl1 gene abundance within septic tanks in Thailand

Antimicrobial resistance (AMR) poses serious global public health threat and wastewater treatment (WWT), including septic tanks, are a significant source of AMR genes to the environment. Environmental monitoring of broad-range AMR genes remains a challenge. The class-1 integron-integrase (intI1) gene has been proposed as a proxy for overall AMR abundance, but there is no consensus on the qPCR primer set to use. A systematic review of the literature found 65 primer sets. The coverage and specificity of each, including newly designed MGB-TaqMan primer-probe, was evaluated against a database of clinical and environmental intl1, intl1-like and non-intl1 sequences. Three primer sets were selected, laboratory validated for DNA and mRNA quantification and used to quantity intl1 gene abundance from household and healthcare conventional septic tanks (CST) and novel household Solar Septic Tanks (SST) in Thailand. Specificity of the intl1 septic tank amplicons showed that no primer set could distinguish between intl1 and intl-1 like sequences. Each primer set showed the same trends across septic tanks, with highest gene abundance in influent>sludge>effluent. There was no statistical difference between the same sample quantified by the three primer sets. However, when comparing gene abundances from the same primer set across septic tanks, statistical differences between different sample types were observed for one primer set but not the others. This may lead to different interpretation of risk associated with each reactor in spreading intl1 to the environment. Comparing reactor types, intI1 abundance in the effluent was lowest in the SST-household<CST-household<CST-healthcare. Depending on primer set used, 31 to 42% of intI1 was removed from effluent of the CST-household tank with accessible influent. Our study provided insight into the importance of intl1 primer choice. We propose the use of the validated set (F3-R3) for optimal intI1 quantification and towards the goal of achieving standardisation across environmental studies.

molecular biology↗

bioRxiv2022

Ecological Observations Based on Functional Gene Sequencing Are Sensitive to the Amplicon Processing Method

Until recently, the de-facto method for short read-based amplicons reconstruction is a sequence similarity threshold approach (Operational taxonomic Units OTUs). This assumption was relaxed by shifting to Amplicon Sequencing Variants (ASVs) where distributions are fitted to abundance profiles of individual genes using a noise-error model. Whilst OTUs-based approach is still useful for 16SrRNA/18S rRNA regions, where typically 97-99% thresholds are used, their utility to functional genes is still debatable as there is no consensus on how to cluster the sequences together. Here, we compare OTUs- and ASVs-based reconstruction approaches as well as taxonomy assignment methods, Naive Bayesian Classifier (NBC) and Bayesian Lowest Common Ancestor Algorithm (BLCA), using functional genes dataset from the microbial nitrogen-cycling community in the Brouage mudflat (France). A range of OTU similarity thresholds and ASV were used to compare amoA (AOA and AOB), nxrB, nirS, nirK and nrfA communities between differing sedimentary structures. We show that for AOA-amoA and nrfA, the use of ASV led to differences in the communities between sedimentary structures whereas the use of OTUs didnt. Conversely, significant differences were detected when using OTU (97%) for AOB-amoA but not with ASV or OTUs at other similarity thresholds. Interestingly, conclusions drawn from the other three functional genes were consistent between amplicon reconstruction methods. We also show that, when the sequences in the reference-database are related to the environment in question, BLCA leads to more phylogenetically relevant classifications. However, when the reference database contains sequences more dissimilar to the ones retrieved, NBC helps obtain more information. ImportanceSeveral analysis pipelines are available to microbial ecologists to process amplicon sequencing data yet to-date, there is no consensus as to the most appropriate method, and it becomes more difficult for genes that encode a specific function (functional genes). Standardised approaches need to be adopted to increase reliability and reproducibility of environmental amplicon sequencing-based datasets. In this paper, we argue that the recently developed ASV approach offers a better opportunity to achieve such standardisation compared to OTUs for functional genes. We also propose a comprehensive framework for quality filtering of the sequencing reads based on protein sequence verification and merging.

bioinformatics↗

bioRxiv2020

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

RT-Q-PCR, and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same sample. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript diversities (i.e. OTU counts within a sample) can be biased by the RT, the comparison of {beta} diversities (i.e. differences in OTU counts between samples) is reliable as those biases are reproducible between environments. Originality-Significance StatementIs the complementary DNA (cDNA) produced after Reverse Transcription (RT) a faithful representation of the starting RNA? This is a fundamental and important question for transcriptomic-based studies in environmental microbiology that aim to quantify and/or examine the diversity of transcripts via RT approaches. Yet little is known about the reliability and reproducibility of this step. Here, we evaluated the effect of the two main components of the RT reaction - the retro transcriptase enzyme and priming strategy (gene specific vs random priming), on the quantification and diversity of cDNA. We found that both have a significant impact. We further provide evidence to enable informed choices as to the enzyme and priming combinations to improve the performance of RT-PCR approaches. Taken together, this work will improve the reliability and reproducibility of transcript-based studies in environmental microbiology.

molecular biology↗

bioRxiv2018

Differential amplicons for the evaluation of RNA integrity extracted from complex environmental samples

BackgroundReliability and reproducibility of transcriptomics-based studies are highly dependent on the integrity of RNA. Microfluidics-based techniques based on ribosomal RNA such as the RNA Integrity Number (RIN) are currently the only approaches to evaluate RNA integrity. However, it is not known if ribosomal RNA reflects the integrity of the meaningful part of the sample, the mRNA. Here we test this assumption and present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of long to short RT-Q-PCR amplicons of the glutamine synthetase A (glnA) transcript.\n\nResultsWe successfully designed and tested two Ramp indexes targeting glnA transcripts. We showed in a suite of experimental degradations of RNA extracted from sediment that while the RIN in general did reflect the degradation status of the RNA well the Ramp mapped mRNA degradation better as reflected by changes in Reverse Transcriptase Quantitative PCR (RT-Q-PCR) results. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of the 16S rRNA, amoA and glnA transcript which was successful even form the highly-degraded samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed between successively degraded 16S rRNA transcripts profiles.\n\nConclusionAs demonstrated, transcripts can be quantified and sequenced even from highly degraded samples. Therefore, we strongly recommend that a quality check of RNA is conducted to ensure validity of results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.

microbiology↗