ArticleBMC microbiology2021
ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples.
Article in BMC microbiology, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Modified percutaneous vertebral body-intervertebral disc biopsy combined with genetic testing for the diagnosis of early-stage pyogenic spondylodiscitis: a retrospective cohort study.European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society · 2026Article
- A framework for optimising arthropod DNA quality and quantity for modern sequencing tools using hard ticks (Ixodidae).Parasitology research · 2026Article
- Methodological bias shapes the interpretation of the urinary microbiome in low-biomass systems.Frontiers in microbiology · 2026Review
- Article
- Comparison of RNA- and DNA-based 16S amplicon sequencing to find the optimal approach for the analysis of the uterine microbiome.Scientific reports · 2025Article
- The infant microbiota hopscotches between community states toward maturation-longitudinal stool parameters and microbiota development in a cohort of European toddlers.ISME communications · 2025Article
- Insights intoMicroorganisms · 2024Article
- Long journey of 16S rRNA-amplicon sequencing toward cell-based functional bacterial microbiota characterization.iMetaOmics · 2024Review
- Definitions of Dysbiosis in Chronic Lung Allograft Dysfunction and High Bacterial Biomass.American journal of respiratory and critical care medicine · 2024Article
- Reliability of Droplet Digital PCR Alone and in Combination with Interleukin-6 and Procalcitonin for Prognosis of Bloodstream Infection.Infection and drug resistance · 2024Article
- Contribution of the Oral and Gastrointestinal Microbiomes to Bloodstream Infections in Leukemia Patients.Microbiology spectrum · 2023Article
- Fast and efficient template-mediated synthesis of genetic variants.Nature methods · 2023Article
- Genome-wide mapping of gene-microbe interactions in the murine lung microbiota based on quantitative microbial profiling.Animal microbiome · 2023Article
- Artificial Intelligence in microbiomes analysis: A review of applications in dermatology.Frontiers in microbiology · 2023Review
- Skin microbiota analysis in patients with anorexia nervosa and healthy-weight controls reveals microbial indicators of healthy weight and associations with the antimicrobial peptide psoriasin.Scientific reports · 2022Article
Corrections and comments
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundOne limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.
resultsUsing a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts.
conclusionOur improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.
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