Chapter Four · failure evidence
What Amplicon Sequencing & Metagenomics got wrong, from 35 dissertations
Across the doctoral research records, amplicon sequencing and metagenomic profiling frequently encountered limitations in taxonomic resolution, sample biomass, and computational analysis. Researchers also reported critical failures involving off-target amplification artifacts, reagent contamination, and the high cost or assembly difficulties of shotgun metagenomics. These records come from PhD theses at 17 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Targeted 16S rRNA amplicon sequencing fails to provide sufficient taxonomic and functional resolution
Researchers rejected or failed with 16S rRNA amplicon sequencing because it could not resolve closely related species, recent speciation events, or strain-specific functional traits. The approach also suffered from gene copy number heterogeneity and lacked the sensitivity required to detect low-abundance pathogen targets in complex tissue microbiomes.
Considered and rejected
Considered and rejected: Rejected using 16S rRNA gene amplicon sequencing due to copy number heterogeneity and poor resolution for distinguishing closely related rhizobial species.
Harnessing the Power of The Endemic Soil Microbiome for Developing Tailored Crop Management · Texas Tech
Considered and rejected
Considered and rejected: Considered amplicon (16S rRNA) sequencing but decided against it in favor of shotgun metagenomic sequencing (SMS) due to SMS providing higher resolution and strain-specific functional information with less bias.
Alteration of the gut microbiome by scutellaria-coptis herb couple and metformin in type 2 diabetes · OpenBU
Considered and rejected
Considered and rejected: Single-gene marker 16S rRNA direct amplicon sequencing from plant tissue was rejected for disease diagnostics due to low taxonomic resolution.
Bacterial Plant Pathogen Identification using Genomics and Metagenomics · Virginia Tech
Tried and failed
16S rRNA gene amplicon sequencing applied to detecting low-abundance specific bacterial pathogens. Outcome: no signal. Reason: insufficient sensitivity or taxonomic resolution to detect low-abundance pathogen targets within complex tissue microbiomes
Considered and rejected
Considered and rejected: Single 16S rRNA gene amplicon sequencing alone was deemed insufficient to detect functional shifts or plasmid-borne virulence genes in shrimp gut microbiome studies.
Considered and rejected
Considered and rejected: Rejected using 16S rRNA V4 and V1-V3 amplicon markers to distinguish Prevotella species in clinical/microbiome cohorts due to lack of taxonomic resolution and erroneous clade placement compared to core-genome phylogenetics.
Considered and rejected
Considered and rejected: Rejected relying on 16S rRNA gene amplicon profiling because identical V4 regions fail to resolve recent speciation in Pseudomonas
Phylogeny, pangenomics, and predicted functional diversity of maize rhizosphere Pseudomonas · Publikationssystem UB Tuebingen
Low biomass, poor sequencing depth, and reagent contamination cause sample dropouts
DNA extraction from low-biomass environmental and tissue samples frequently yielded poor PCR amplification, inadequate read depth, and low genome coverage breadth compared to higher-quality samples. In addition, commercial extraction kit contamination and non-correlating contaminant DNA proportions confounded microbial abundance profiling and forced sample exclusions.
Tried and failed
estimating relative microbial abundance from contaminant DNA applied to microbiome taxon abundance profiling. Outcome: no signal. Reason: contaminant DNA taxa proportions did not correlate with relative abundances in 16S rRNA sequencing
COMPUTATIONAL EXPLORATION OF THE GENETIC FACTORS BEHIND TRANSCRIPTIONAL REGULATION · Cornell
Tried and failed
amplicon sequencing of environmental DNA applied to deep subsoil samples. Outcome: no signal. Reason: insufficient DNA yield or quality from deep soil horizons prevented generation of usable sequencing reads
Lost to a baseline
Microbiome samples excluded from sequencing analysis due to poor sequence quality and insufficient read depth.
Tried and failed
16S rRNA amplicon sequencing applied to low-biomass filter media DNA extracts. Outcome: no signal. Reason: samples exhibited very low to no PCR amplification during quality control
Upstream Effects on Microbial Community Selection in Biological Activated Carbon Filters · unevada
Tried and failed
commercial kit-based DNA extraction applied to low-biomass host microbiome profiling. Reason: extraction kit reagents were contaminated with Mollicutes, confounding sequencing results and requiring data removal
COMMUNITY ASSEMBLY IN HOST-ASSOCIATED AND ENVIRONMENTAL MICROBIOMES · Georgia Tech
Lost to a baseline
Clinical saliva sequencing genome coverage breadth outperformed sewage amplicon sequencing (mean 91.8% in saliva vs 22.6% in sewage).
Advancing wastewater-based epidemiology: Engineered solutions for efficient sampling and RNA preservation of SARS-CoV-2 · Georgia Tech
Lost to a baseline
Three PCR replicate samples (all Siderastrea siderea samples from the lagoon) yielded relatively few reads and were excluded from subsequent microbiome analyses.
Statistical imputation, differential abundance testing, and machine learning models fail on microbiome count data
Missing data imputation distorted ecological diversity distributions, and conservative differential abundance tests failed to retain significant taxa following false discovery rate correction. Furthermore, alpha diversity failed to correlate with absolute abundance, while transfer learning and shallow embedding models failed to beat simple baselines or random classification.
Tried and failed
statistical and curve-fitting missing data imputation applied to sparse microbiome abundance data. Reason: significantly distorted ecological diversity distributions compared to real data
Deep Learning for Enhancing Human and Environmental Health · Virginia Tech
Tried and failed
compositional differential abundance testing using ALDEx2 applied to soil microbiome taxonomic count data. Outcome: no signal. Reason: ALDEx2 was overly conservative compared to other differential abundance methods, failing to detect significant OTU shifts
MICROBIAL NITROGEN IMMOBILIZATION AS A TOOL TO MANAGE WEEDS IN AGROECOSYSTEMS · Cornell
Tried and failed
correlating alpha diversity with total microbial abundance applied to host-associated microbiome profiling. Outcome: no signal. Reason: diversity metrics failed to correlate with absolute abundance in samples dominated by specific taxa
Exploration of the chemodiversity and microbial composition in marine sponges · Georgia Tech
Tried and failed
16S rRNA amplicon sequencing differential abundance testing applied to low-biomass tissue microbiome samples. Outcome: no signal. Reason: Candidate differentially abundant taxa did not remain statistically significant after false discovery rate correction
Microbes of the female genital tract, inflammation and ovarian cancer · Imperial
Tried and failed
summed shallow embeddings for sample classification applied to microbiome abundance fold-change profiles. Outcome: worse than baseline. Reason: summed embedding vectors failed to outperform random classification benchmarks
Disease2Vec: a method of determining disease from gut microbiome using neural embeddings · Harvard
Lost to a baseline
In CRC microbiome classification with projection weighting (Study 2 as target), Trans-LDA achieved an AUC of 0.4626, performing worse than single-sample LDA baseline (AUC 0.4869).
TRANSFER LEARNING IN CLASSIFICATION AND REGRESSION WITH SUMMARY STATISTICS · Penn
Primer non-specificity and enzyme selection introduce severe amplification artifacts and bias
High-order multiplexing and targeted pathogen primers suffered from massive off-target binding to background DNA, generating severe poly-A and poly-T artifacts or poor on-target mapping. Substituting high-fidelity polymerases or domain-specific primer designs also resulted in steep drops in sequencing depth and lower observed diversity compared to standard universal primers.
Tried and failed
high-order multiplex amplicon sequencing applied to environmental microbiome profiling. Reason: massive off-target amplification producing poly-A and poly-T sequencing artifacts that dominated read output
Characterization of environmental microbiomes impacted by Iowa agriculture · Iowa State
Tried and failed
domain-specific targeted primers for amplicon sequencing applied to sediment microbial community diversity profiling. Outcome: worse than baseline. Reason: produced fewer amplicon sequence variants and lower diversity estimates than universal prokaryotic primers
COMMUNITY ASSEMBLY IN HOST-ASSOCIATED AND ENVIRONMENTAL MICROBIOMES · Georgia Tech
Tried and failed
amplicon sequencing targeting specific pathogen genomes applied to complex environmental microbiome samples. Outcome: no signal. Reason: massive off-target primer binding to background bacterial DNA caused poor on-target mapping
Advancing wastewater-based epidemiology: Engineered solutions for efficient sampling and RNA preservation of SARS-CoV-2 · Georgia Tech
Lost to a baseline
High-fidelity Q5 polymerase reduced sequencing depth by >100-fold compared to standard Taq/AmpliTaq Gold polymerases, severely reducing variant calling sensitivity in amplicon sequencing.
The Study of the Environmental Etiologies of Urothelial Bladder Cancers through Mutational Analysis · JScholarship
Tried and failed
synthetic spike-in oligo library for contamination tracking applied to multiplexed viral amplicon sequencing. Reason: candidates suffered poor amplification, overlap with contaminants, or synthesis and preparation impurities
Shotgun metagenomics is impeded by host contamination, assembly failures, and high sequencing costs
Shotgun metagenomic pipelines were rejected or failed when overwhelming host DNA contamination biased profiles or when binning pipelines could not yield metagenome-assembled genomes meeting completeness thresholds. Additionally, whole-genome metagenomic sequencing was considered but rejected because its high cost made it prohibitively expensive compared to amplicon sequencing.
Considered and rejected
Considered and rejected: Rejected WIMP metagenomic pipeline for patient microbiota profiling in favor of 16S amplicon sequencing due to overwhelming host DNA contamination and Eschericia bias.
The Microbiota of Pressure Injuries: Antibiotic Tolerance and Bacterial Dynamics in Polymicrobial Biofilm Infections and Chronic Wound Patients · Carleton University Institutional Repository
Considered and rejected
Considered and rejected: Rejected whole-genome metagenomic sequencing in favor of 16S rRNA gene amplicon sequencing due to whole-genome sequencing being prohibitively expensive.
Soil microbial community resistance and resilience to biodiesels vs. petroleum diesel · Texas Tech
Tried and failed
standard metagenomic binning and assembly pipelines applied to gut microbiome metagenomic data. Outcome: data insufficient. Reason: failed to yield metagenome-assembled genomes meeting standard completeness and contamination thresholds
Segatella clades adopt distinct roles within a single individual’s gut · Cornell
Left open by the authors
Problems the authors named and did not get to.
Left open
Perform amplicon sequencing on 2017 and 2018 Dyngjusandur samples to evaluate temporal microbial community composition across sites. Blocker: Requires access to physical soil samples from 2017/2018 and a molecular biology wet lab.
Distribution, Characterization, & Temporal Study of Biosignatures at the Dyngjusandur, Iceland Mars Analog · Georgia Tech
Left open
Improve DARTE-QM primer designs and experimentally benchmark performance against alternative microbiome quantification methods. Blocker: Requires wet lab access, primer synthesis, sequencing platforms, and environmental microbiome samples
Characterization of environmental microbiomes impacted by Iowa agriculture · Iowa State
Left open
Conduct experimental studies on host-depleted blood samples to verify the presence of a genuine cancer blood microbiome. Blocker: Requires biological blood samples, wet lab host-depletion protocols, and physical sequencing facilities
MICROBIOTA SIGNATURES OF BLOOD AND TUMOURS WITH METAGENOMIC APPROACHES · DalSpace
Left open
Incorporate 16S rRNA and nifH amplicon sequencing datasets to assess wastewater eutrophication effects on marine diazotrophs. Blocker: Requires specific 16S rRNA and nifH amplicon sequencing datasets or wet-lab sample collection and sequencing from wastewater-impacted marine sites
Effects of Shipping Traffic On Marine Microbial Community Composition · DalSpace
Left open
Update predictive metagenomics databases with bai operon annotations to predict site-specific bile acid dehydroxylation differences from mucosal/faecal 16S sequencing data. Blocker: Requires the private patient 16S amplicon sequencing dataset generated in the thesis to predict cohort site-specific differences
Comparative analysis of sporadic and colitis associated cancers · Imperial
Left open
Perform taxonomic 16S rRNA sequencing to confirm gut microbiome colonization and persistence in microbiome transfer recipient mice. Blocker: Requires wet lab access, mouse models, fecal sample collection, and 16S rRNA sequencing facilities.
Effects of moderate alcohol consumption in a murine model of multiple sclerosis · UT Austin
Left open
Perform 16S rRNA gene amplicon sequencing on increasing river biofilm subsamples to generate species saturation and rarefaction curves. Blocker: Requires wet-lab biofilm sample collection and 16S rRNA gene amplicon sequencing.
Insights into antimicrobial resistance patterns in Lower Austrian rivers along gradients of faecal pollution Untersuchung von Antibiotikaresistenzmustern in niederösterreichischen · DSpace-CRIS at TU Wien
Left open
Perform targeted amplicon sequencing of loss-of-function loci from patient samples to evaluate intra-host pathogen diversity in infection and transmission. Blocker: Requires clinical patient samples and wet-lab targeted amplicon sequencing capabilities
Left open
Collect stool samples and perform shotgun metagenomic sequencing to profile gut microbiome composition in individuals with public speaking anxiety. Blocker: Requires collecting human biological stool samples and performing wet-lab metagenomic sequencing
Assessment of diet in relation to public speaking anxiety · Harvard
Left open
Perform faecal microbiome sequencing and metagenomic profiling to link microbial taxonomy with SCFA and bile acid metabolomic profiles. Blocker: Requires wet lab faecal sample collection, DNA extraction, and metagenomic sequencing.
Investigating the role of different dietary patterns on gut metabolites · Imperial
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