ArticlemBio2021
Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers.
Article in mBio, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 32 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
32 citing papers in PubMed, 39 citations in OpenAlex.
- Concordance of molecular microbiology and conventional culture techniques for infected diabetic foot ulcer management.Diabetic medicine : a journal of the British Diabetic Association · 2025Trial
- The role of the respiratory microbiome in acute and chronic respiratory diseases.Nature reviews. Microbiology · 2026Review
- Bile Microbiota Profiling in Obese and Non-Obese Patients: A Comparison of Shotgun Metagenomics and 16S rRNA Amplicon Sequencing.Life (Basel, Switzerland) · 2026Article
- An ode to the 16S rRNA gene: its history, importance, caveats and future in microbiome research.Microbial genomics · 2026Review
- Microbial Detection in Community-Acquired Pleural Space Infections Using the BIOFIRE Joint Infection Panel and Individual Polymerase Chain Reaction Assays.Clinical infectious diseases : an official publication of the Infectious Diseases Society of America · 2026Article
- Odontogenic Brain Abscess in a Hereditary Haemorrhagic Telangiectasia (HHT) Patient: Case Report with a Comprehensive Literature Review.Tropical medicine and infectious disease · 2026Article
- Critical assessment of intratumor and low-biomass microbiome using long-read sequencing.bioRxiv : the preprint server for biology · 2026Article
- Detection of Pathogens by a Novel User-Developed Broad-Range BR 16S PCR rRNA Polymerase Chain Reaction/Gene Sequencing Assay: Multiyear Experience in a Large Canadian Healthcare Zone.Microorganisms · 2026Article
- Presence of bacterial DNA in synovial fluid from the temporomandibular joint in patients with temporomandibular joint disorders.Frontiers in oral health · 2026Article
- Intratumoral bacteria are immunosuppressive and promote immunotherapy resistance in head and neck squamous cell carcinoma.Nature cancer · 2026Article
- The triad of maternal gut-breast milk-infant gut microbial transmission in early life as a critical pathway for microbial inheritance.Gut microbes · 2025Article
- Quantitative profiling of bacterial communities via full length 16S rRNA gene sequencing with internal controls: optimization and validation across diverse human microbiomes.BMC microbiology · 2025Article
- Rapid and reliable species-level identification from clinical samples using 16 S rRNA gene nanopore sequencing analysis.Scientific reports · 2025Article
- Nationwide multicentre study of Nanopore long-read sequencing for 16S rRNA-species identification.European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology · 2025Article
- CleanSeqU algorithm for decontamination of catheterized urine 16S rRNA sequencing data.Scientific reports · 2025Article
- Gut microbiota-derived extracellular vesicles form a distinct entity from gut microbiota.mSystems · 2025Article
- 16S Ribosomal RNA Gene PCR and Sequencing for Pediatric Infection Diagnosis, United States, 2020-2023.Emerging infectious diseases · 2025Article
- A retrospective study on blood microbiota as a marker for cognitive decline: implications for detecting Alzheimer's disease and amnestic mild cognitive impairment in Republic of Korea.Osong public health and research perspectives · 2025Article
- Evaluating the feasibility, sensitivity, and specificity of next-generation molecular methods for pleural infection diagnosis.Microbiology spectrum · 2025Observational
- State-of-the-art approaches in the investigation of human seminal bacteriome using metagenomic methods.Frontiers in reproductive health · 2025Review
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
In this article, we investigate patterns of microbial DNA contamination in targeted 16S rRNA amplicon sequencing (16S deep sequencing) and demonstrate how this can be used to filter background bacterial DNA in diagnostic microbiology. We also investigate the importance of sequencing depth. We first determined the patterns of contamination by performing repeat 16S deep sequencing of negative and positive extraction controls. This process identified a few bacterial species dominating across all replicates but also a high intersample variability among low abundance contaminant species in replicates split before PCR amplification. Replicates split after PCR amplification yielded almost identical sequencing results. On the basis of these observations, we suggest using the abundance of the most dominant contaminant species to define a threshold in each clinical sample from where identifications with lower abundances possibly represent contamination. We evaluated this approach by sequencing of a diluted, staggered mock community and of bile samples from 41 patients with acute cholangitis and noninfectious bile duct stenosis. All clinical samples were sequenced twice using different sequencing depths. We were able to demonstrate the following: (i) The high intersample variability between sequencing replicates is caused by events occurring before or during the PCR amplification step. (ii) Knowledge about the most dominant contaminant species can be used to establish sample-specific cutoffs for reliable identifications. (iii) Below the level of the most abundant contaminant, it rapidly becomes very demanding to reliably discriminate between background and true findings. (iv) Adequate sequencing depth can be claimed only when the analysis also picks up background contamination.
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.