ArticlePloS one2012
High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers, 1 of them a synthesis that pooled it.
What it found
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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
45 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Pooled it
- Composition of gut microbiota in infants in China and global comparison.Scientific reports · 2016Trial
- Bifidobacterial succession and correlation networks in a large unselected cohort of mothers and their children.Applied and environmental microbiology · 2013Trial
- A Refined Method for Fecal Microbiota Transplantation Using Nutella.Clinical and translational science · 2026Article
- Effects of repeated freeze and thaw cycles on the stability of faecal microbiome composition.Scientific reports · 2026Article
- Article
- Next-generation sequencing applications in food science: fundamentals and recent advances.Frontiers in bioengineering and biotechnology · 2025Review
- Bacterial Community Composition and Isolation of Actinobacteria from the Soil of Flaming Mountain in Xinjiang, China.Microorganisms · 2023Article
- Histopathological Findings and Metagenomic Analysis of Esophageal Papillary Proliferation Identified in Laying Broiler Breeders.Veterinary sciences · 2022Article
- Capturing marine microbiomes and environmental DNA: A field sampling guide.Frontiers in microbiology · 2022Review
- Advantage of 16S rRNA amplicon sequencing in Helicobacter pylori diagnosis.Helicobacter · 2021Article
- Procedures for Fecal Microbiota Transplantation in Murine Microbiome Studies.Frontiers in cellular and infection microbiology · 2021Review
- Toward Best Practice in Livestock Microbiota Research: A Comprehensive Comparison of Sample Storage and DNA Extraction Strategies.Frontiers in microbiology · 2021Article
- Impact of host genetics on gut microbiome: Take-home lessons from human and mouse studies.Animal models and experimental medicine · 2020Review
- The rodent vaginal microbiome across the estrous cycle and the effect of genital nerve electrical stimulation.PloS one · 2020Article
- Influence of 16S rRNA variable region on perceived diversity of marine microbial communities of the Northern North Atlantic.FEMS microbiology letters · 2019Article
- A 16S rDNA PCR-based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information.BMC microbiology · 2019Article
- Considerations When Designing a Microbiome Study: Implications for Nursing Science.Biological research for nursing · 2019Article
- Community profiling of the urinary microbiota: considerations for low-biomass samples.Nature reviews. Urology · 2018Review
- Cross-Reactivity of Prokaryotic 16S rDNA-Specific Primers to Eukaryotic DNA: Mistaken Microbial Community Profiling in Environmental Samples.Current microbiology · 2018Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
5 authors.
Funding
Abstract
The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.
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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.