Library · Biology & lab methods
What Amplicon Sequencing & Metagenomics got wrong, from 24 theses
Summary written by a model from the records below: The records document practical bottlenecks in amplicon sequencing and metagenomic profiling across environmental, clinical, and host-associated systems. Failures frequently stemmed from insufficient taxonomic resolution, severe off-target amplification, sample biomass limitations, reagent contamination, and computational modeling deficiencies. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and 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.
Plus 10 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Marker gene amplicon sequencing fails to provide sufficient taxonomic resolution or functional information
Researchers frequently rejected or failed with 16S rRNA amplicon sequencing because marker genes cannot distinguish closely related species, resolve recent speciation, or detect low-abundance pathogens. In addition, 16S rRNA gene profiling suffers from copy number heterogeneity and cannot identify plasmid-borne virulence genes or functional shifts.
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 · PhD
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 · Boston University · Master's
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 · Doctorate
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.
The Influence of Prebiotics, Probiotics, and Exposure to an Opportunistic Pathogen on the Intestinal Microbiome of White Shrimp (Litopenaeus vannamei) · Virginia Tech · Master's
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 · Tübingen · PhD
Non-specific primer binding and enzyme selection cause severe amplification artifacts
Targeted and multiplex amplicon designs suffered from extensive off-target amplification and binding to background DNA, producing artifacts such as poly-A and poly-T reads. Furthermore, alternative primers and high-fidelity polymerases yielded poorer diversity estimates or drastically lower sequencing depth compared to standard approaches.
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 · PhD
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 · PhD
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 · PhD
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 · Johns Hopkins · PhD
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
Interrogation of Viral Genomes, Viral Translation, and Intellectual Property Systems for Access to Essential Research Tools · Harvard · PhD
Low biomass and challenging environmental matrices cause sample dropout and read depletion
Difficult environmental samples such as deep subsoil and filter media failed to yield adequate DNA quality or PCR amplification for sequencing. Poor sequence quality and low read depth ultimately forced the exclusion of affected samples and replicates from downstream microbiome analyses.
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
CARBON STORAGE AND CYCLING IN AFRICAN DARK EARTHS · Penn · PhD
Lost to a baseline
Microbiome samples excluded from sequencing analysis due to poor sequence quality and insufficient read depth.
EVALUATING THE EFFECTS OF BENZALKONIUM CHLORIDE (BAC) ON EARLY-LIFE STAGE AND JUVENILE RAINBOW TROUT (ONCORHYNCHUS MYKISS) · Saskatchewan · Master's
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 · University of Nevada, Reno · Doctorate
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.
Variation in phenotype and microbiome of three widespread Caribbean corals between mangrove and reef habitats · Boston University · Master's
Contaminant DNA and host material confound microbial profiling
Contaminant taxa in commercial extraction reagents or background DNA distorted microbial profiles and prevented accurate relative abundance estimation. Similarly, overwhelming host DNA contamination and pipeline biases led researchers to abandon metagenomic approaches in favor of targeted profiling.
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 · PhD
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 · Master's
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 · PhD
Statistical correction and imputation methods distort microbiome distributions or lose power
Imputing missing values in sparse microbiome count tables distorted natural ecological diversity distributions. Moreover, conservative differential abundance testing methods and false discovery rate adjustments failed to detect significant taxonomic shifts across conditions.
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 · Doctorate
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 · PhD
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 · Doctorate
Advanced binning and machine learning pipelines fail to outperform standard benchmarks
Standard metagenomic assembly and binning workflows failed to produce metagenome-assembled genomes meeting standard completeness and contamination thresholds. Additionally, transfer learning and shallow embedding models failed to surpass baseline classifiers or random benchmarks on microbiome abundance data.
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 · Master's
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 · PhD
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 · PhD
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 · PhD
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 · PhD
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 · Dalhousie · Master's
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 · Dalhousie · Master's
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 · PhD
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 · PhD
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.
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
Genome-wide association identifies antibiotic resistance and susceptibility mutations in Neisseria gonorrhoeae · Harvard · PhD
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 · Master's
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 · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Ana Gabriel Clavere Graciette · Georgia Tech · PhD · 2021
COMMUNITY ASSEMBLY IN HOST-ASSOCIATED AND ENVIRONMENTAL MICROBIOMES - Schuyler Smith · Iowa State · PhD · 2021
Characterization of environmental microbiomes impacted by Iowa agriculture - Brayan Diddey Montoya Torres · Texas Tech · PhD · 2026
From Abattoir to Animal Model: 16S rRNA gene Microbial Profiling of Beef Cattle Lymph Nodes vs. Digesta and Functional Microbiome Changes in C3H/HeJ Mice on High-Fat Casein Diets ± Ammonium-Hydroxide - Joung Min Choi · Virginia Tech · Doctorate · 2026
Deep Learning for Enhancing Human and Environmental Health - Jerusa Brignardello · Imperial · PhD · 2025
Investigating the role of different dietary patterns on gut metabolites - Evan Arron Spencer Kohlman · Saskatchewan · Master's · 2025
EVALUATING THE EFFECTS OF BENZALKONIUM CHLORIDE (BAC) ON EARLY-LIFE STAGE AND JUVENILE RAINBOW TROUT (ONCORHYNCHUS MYKISS) - Kira Noelle Allison · Carleton University · Master's · 2025
The Microbiota of Pressure Injuries: Antibiotic Tolerance and Bacterial Dynamics in Polymicrobial Biofilm Infections and Chronic Wound Patients - Brian Beardsall · Dalhousie · Master's · 2025
Effects of Shipping Traffic On Marine Microbial Community Composition - Xieyue Xiao · Cornell · PhD · 2024
Segatella clades adopt distinct roles within a single individual’s gut - Duncan Roberts · Imperial · PhD · 2024
Comparative analysis of sporadic and colitis associated cancers - Gyuhyon Cha · Georgia Tech · PhD · 2024
Advancing wastewater-based epidemiology: Engineered solutions for efficient sampling and RNA preservation of SARS-CoV-2 - Jessica Lynn Sutter · Tübingen · PhD · 2024
Phylogeny, pangenomics, and predicted functional diversity of maize rhizosphere Pseudomonas
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