ArticleMicrobiologyOpen2020
Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.
Article in MicrobiologyOpen, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 49 papers.
What it found
Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.
The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
49 citing papers in PubMed, 112 citations in OpenAlex.
- Article
- Amplification Efficiency of Quantitative PCR Reactions is Improved by Addition of Non-Target DNA.Microbial ecology · 2026Article
- Emerging Therapeutic Approaches for Modulating the Intestinal Microbiota.Pharmaceutics · 2026Review
- Comparison of ESKAPE Pathogen Levels in Wastewater and Receiving Water Bodies Using Agar-Based Enumeration and Real-Time PCR.Current microbiology · 2026Article
- Human pluripotent stem cell-derived skin organoids enabled pathophysiological model of Mycobacterium tuberculosis infection.Nature communications · 2025Article
- Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR.Nature protocols · 2025Review
- State-Specific Extraction of Environmental DNA: Spike-and-Recovery Controls to Validate and Optimise Extraction Protocols.Environmental microbiology · 2025Article
- Challenges and Opportunities in Analyzing Cancer-Associated Microbiomes.Cancer research · 2025Review
- Observation Bias in Metabarcoding.Molecular ecology resources · 2025Article
- Uncertainty Modeling Outperforms Machine Learning for Microbiome Data Analysis.bioRxiv : the preprint server for biology · 2025Article
- Article
- Nested PCR to optimizeMicrobiology spectrum · 2025Article
- Development of a quantitative PMA-16S rRNA gene sequencing workflow for absolute abundance measurements of seawater microbial communities.Environmental microbiome · 2025Article
- Secretory IgM regulates gut microbiota homeostasis and metabolism.Nature microbiology · 2025Article
- Tracking Different States of Spiked Environmental DNA Using Multiplex Digital PCR Assays.Environmental microbiology · 2025Article
- The quest for environmental analytical microbiology: absolute quantitative microbiome using cellular internal standards.Microbiome · 2025Review
- Advancing time-since-interval estimation for clandestine graves: a forensic ecogenomics perspective into burial and translocation timelines using massively parallel sequencing.Frontiers in microbiology · 2025Review
- Categorizing and characterizing intestinal dysbiosis: evaluating stool microbial test clinical utility.Frontiers in microbiomes · 2025Article
- Drivers of antimicrobial resistance in pig production systems of Uganda.Communications earth & environment · 2025Article
- A comprehensive comparison of web-based tools for amplicon-metagenomic analysis.Frontiers in microbiology · 2025Review
Corrections and comments
- Erratum issuedCORRIGENDUM.2022
Authors and funding
8 authors at 4 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.
Indexed as
Identifiers
What OpenQuestion holds
Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.