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What Bioinformatics Homology & Sequence Alignment got wrong, from 49 theses
Summary written by a model from the records below: Sequence alignment and homology mining methods frequently struggle when applied to divergent biological sequences, complex domain architectures, and large-scale genomic datasets. Computational researchers often find that specialized machine learning models and ab initio predictors underperform established homology baselines, while trimming strategies and locus selection choices can compromise downstream phylogenetic inference. 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 9 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
High sequence divergence and evolutionary distance cause alignment and homology detection failure
Standard pairwise alignments, orthology tools, and read mappers fail to find matches when sequence divergence is high or pairwise identity drops below 15 percent. Researchers frequently discard direct pairwise sequence searches in favor of profile hidden Markov models, structural homology, or truncation assays to detect distant relationships.
Tried and failed
reference-based read alignment to consensus genomes applied to highly diverse viral single-cell RNA. Outcome: no signal. Reason: extensive sequence diversity and serotypic variation prevented read mapping to the static reference
Single-Cell Biology of Respiratory Viral Infections in the Nasal Mucosa · Harvard · PhD
Tried and failed
standard sequence similarity search applied to divergent protein domain sequence identification. Outcome: no signal. Reason: Pairwise sequence alignment failed to detect highly divergent homologs across distant eukaryotic lineages.
Computational Approaches to Developmental Biology · Harvard · PhD
Tried and failed
strict short-sequence alignment matching applied to CRISPR spacer-to-protospacer virus-host linking. Outcome: no signal. Reason: overly strict alignment thresholds failed to match divergent sequences, requiring relaxed search parameters
Novel microbes and viruses with roles in biogeochemical cycling and eukaryogenesis in marine systems · UT Austin · PhD
Tried and failed
automated orthology inference software applied to distantly related cross-phylum sequence homology. Outcome: no signal. Reason: failed to detect highly divergent homologous immune receptors across deep evolutionary distances
Evolution of Cell Types in Tunicate Blood · Harvard · PhD
Considered and rejected
Considered and rejected: Rejected using sequence homology alone for detecting all viral mimics, acknowledging >70% cannot be detected without structural homology.
Evolutionary History of Immunomodulatory Genes of Giant Viruses · Virginia Tech · Master's
Considered and rejected
Considered and rejected: Aligning GBS reads of Melampsora paradoxa to the Melampsora americana reference genome R15-033-03 was rejected due to high sequence divergence (37.6% SNP profile similarity, 90.8% ITS identity) causing poor read alignment and reduced SNP quality.
Population biology of Melampsora americana and Melampsora paradoxa facilitating host resistance breeding in Salix · Cornell · PhD
Considered and rejected
Considered and rejected: Decided against relying solely on pairwise BLAST searches for viral ortholog identification in favor of profile HMMs of ViPhOGs to overcome sequence divergence.
On Bioinformatics of the Human Gut Virome · Tübingen · PhD
Tried and failed
sequence similarity search for functional annotation applied to candidate divergent loci in non-model genome. Outcome: no signal. Reason: queries failed to produce significant alignment matches in reference databases due to incomplete annotations and sequence divergence
Host-parasite interactions at the genetic level in the Schistocephalus solidus tapeworm, copepods, and threespine stickleback system · UT Austin · PhD
Tried and failed
cross-species reference germline alignment applied to non-model organism antibody repertoire sequencing. Reason: introduced unpredictable sequence alignment biases and annotation errors due to germline divergence
USING SEROLOGY TO UNDERSTAND BAT-VIRUS INTERACTIONS ACROSS SCALES · Cornell · PhD
Tried and failed
conventional sequence alignment algorithms applied to highly diverse protein sequences. Outcome: no signal. Reason: extremely low pairwise sequence identity below 15 percent
UNDERSTANDING THE STRUCTRAL DYNAMICS AND EVOLUTIONARY DIVERSIFICATION OF RIBONUCLEOTIDE REDUCTASES · Cornell · PhD
Considered and rejected
Considered and rejected: Rejected identifying functional CiGnRH1 enhancers by sequence conservation alone across distant ascidians (Phallusia, Halocynthia), switching to systematic truncation assays due to lack of detectable sequence homology.
Evolution and development of the olfactory neurons in the olfactores clade · Oxford · PhD
Inappropriate locus selection or sequence representation degrades phylogenetic resolution
Phylogenetic analyses suffer from poor resolution, tree topology distortion, or long branch attraction when using uninformative marker loci, saturated nucleotides, or unphased ambiguity codes. Investigators reject single-locus markers and raw internal coding sequences in favor of concatenated core operons, codon-based nucleotide alignments, or non-coding terminal repeat sequences.
Considered and rejected
Considered and rejected: Aligning and analyzing raw nucleotide sequences for adenovirus phylogenetic analyses due to nucleotide saturation (translated to amino acid alignment instead).
Phylogeography and Signal Evolution in a Widespread Central American Anole · Harvard · PhD
Considered and rejected
Considered and rejected: Rejected classifying fragmented RefSeq phage beta-prime-like sequences and eukaryotic RdRp within the standard multi-subunit RNAP phylogeny due to severe long branch attraction artifacts
Unraveling the Eco-Evolutionary Complexity of Uncultivated Bacteriophages in the Biosphere · Virginia Tech · Doctorate
Considered and rejected
Considered and rejected: Rejected amino acid alignment-based phylogeny for HCP-3 histone fold domains due to poor phylogenetic resolution; adopted codon-based nucleotide alignment instead.
The mechanism of the peel-1 zeel-1 toxin-antidote system · University of Washington · PhD
Considered and rejected
Considered and rejected: Rejected relying on the HERVH reverse transcriptase (RVT) domain for phylogenetic subtyping due to lower resolution compared to LTR sequence phylogenies
RECOMBINATION-MEDIATED REGULATORY EVOLUTION OF HUMAN ENDOGENOUS RETROVIRUS HERVH · Cornell · PhD
Tried and failed
Bayesian phylogenetic inference using protein sequences applied to highly conserved single-locus protein alignments. Outcome: no signal. Reason: extremely high sequence conservation provided insufficient polymorphic sites to resolve phylogenetic tree topology
Surveillance and prevalence of potential disease in pronghorn (Antilocapra americana) and mule deer (Odocoileus hemionus) in the Texas Panhandle · Texas Tech · Master's
Tried and failed
multilocus sequence typing gene concatenation applied to microbial phylogenetic lineage resolution. Outcome: no signal. Reason: few traditional marker genes lacked sufficient resolution to recover whole-genome phylogenetic structures
Expanding The Bioinformatics Toolbox for Diversity and Taxonomic Studies of Microbial Eukaryotic Pathogens · Georgia Tech · PhD
Lost to a baseline
ParSNP core genome alignment resulted in poor isolate separation (collapsing tree clusters) compared to Roary with RAxML-NG.
Considered and rejected
Considered and rejected: Rejected using nucleotide sequences for deep eukaryote MAPK phylogeny due to poor alignment and high substitution rates outside conserved motifs.
STUDIES OF DEEP HOMOLOGY AND PHENOTYPIC EVOLUTION UNDER A PHYLOGENETIC FRAMEWORK · Johns Hopkins · PhD
Considered and rejected
Considered and rejected: Rejected binary pangenome presence/absence matrix phylogenies in favor of core-genome nucleotide alignments, because accessory gene monophyly was driven merely by 8 gene losses rather than true evolutionary descent.
DEFINING FACTORS IMPORTANT DURING COLONIZATION AND INVASION BY THE PEDIATRIC PATHOGEN KINGELLA KINGAE · Penn · PhD
Tried and failed
phylogenetic reconstruction using internal coding sequences applied to endogenous retrovirus subfamily classification. Outcome: no signal. Reason: coding sequences provided insufficient granularity to resolve subfamilies compared to non-coding terminal repeats
RECOMBINATION-MEDIATED REGULATORY EVOLUTION OF HUMAN ENDOGENOUS RETROVIRUS HERVH · Cornell · PhD
Considered and rejected
Considered and rejected: Rejected using standard unphased IUPAC ambiguity encoding for diploid heterozygous sites because it creates artificial chimeric sequences that distort phylogenetic branch topologies.
Genetic diversity of Candida albicans and Nakaseomyces glabratus across geographic scales and within-host populations · University of Manitoba · PhD
Complex machine learning and ab initio sequence representations fail to beat homology baselines
Pretrained foundation models, neural embeddings, and ab initio structure predictors struggle to generalize across diverse families and frequently lose to homology-based modeling pipelines. These machine learning approaches often suffer from distorted evolutionary encodings, over-reliance on ancestral homology, or artificial benchmark performance on pre-aligned sequences.
Tried and failed
pretrained protein language models for host prediction applied to novel viral variant host classification. Outcome: did not generalise. Reason: models relied on ancestral homology rather than tracking cross-species spillover mutations
Discovering Viral Hosts, Mutations, and Diseases using Machine Learning · Virginia Tech · Doctorate
Lost to a baseline
AlphaFold2 ab initio modelling performed worse on structural alignment to ZmVP14 (RMSD 1.58–1.67 Å) compared to homology modelling with Modeller (RMSD 0.28–0.42 Å).
Drought tolerance and its evolution in conifers including UK commercial species · Oxford · PhD
Lost to a baseline
Ab initio programs ESMFold and trRosetta were outperformed by homology-guided AlphaFold2 across nearly all GFLV proteins in both estimated TM-scores and pLDDT metrics.
THE HOST RESPONSE TO GRAPEVINE FANLEAF VIRUS AND IMPLICATIONS FOR SYMPTOMATOLOGY AND TRANSMISSION · Cornell · PhD
Tried and failed
HMM-based sequence inference preprocessing applied to phenotype prediction from genomic sequences. Outcome: worse than baseline. Reason: Failed to improve out-of-sample performance over BLOSUM matrix inference and degraded performance versus raw sequences
Evaluation of machine learning algorithms for predictive modelling of antibiotic resistance using genomic and spatiotemporal data · Imperial · PhD
Tried and failed
multimodal 3D CNN and transformer sequence embedding applied to remote protein homology retrieval. Outcome: did not generalise. Reason: Learned representation failed to capture cross-family semantic similarity among divergent structural homologs.
Exploring protein biochemistry with deep learning · UT Austin · PhD
Lost to a baseline
AlphaFold2 structural predictions for RhGB01 structural proteins exhibited large poorly supported regions and were outperformed in quality scoring parameters by SWISS-MODEL homology modelling.
Molecular sequence analysis of Crimean-Congo haemorrhagic fever virus and the discovery and characterisation of novel sarbecoviruses · Oxford · PhD
Tried and failed
spatial window evolutionary profiles applied to protein-RNA interface residue prediction. Outcome: worse than baseline. Reason: spatial windows disrupted the linear encoding of evolutionary sequence information
Investigation of sequence and structural characteristics of interfaces in RNA-protein complexes · Iowa State · PhD
Tried and failed
pretrained sequence foundation model embeddings alone applied to nonsense-mediated decay efficiency prediction. Outcome: worse than baseline. Reason: Embeddings lacked explicit positional and mechanistic features captured by simple rule-based heuristics
Learning the language of the genome: AI- and data-driven decoding of genetic variants and disease mechanisms · EPFL · PhD
Multi-domain architectures and structural rearrangements disrupt whole-sequence alignments
Global and full-length alignments fail when proteins or viral genomes contain genomic rearrangements, differing domain architectures, or variable repeat copy numbers. Researchers turn to sliding-window alignments or domain-specific modular analyses to prevent severe residue displacement and avoid masking remote homology.
Tried and failed
direct multiple sequence alignment across distant taxa applied to divergent multi-domain homologous proteins. Outcome: no signal. Reason: mismatched N-terminal domain architectures between distant homologues prevented accurate alignment
Bioinformatic Sequence Analysis Reveals Evolutionary History of Synaptic Genes · Harvard · Master's
Tried and failed
single-round sequence similarity clustering applied to viral genomes with structural rearrangements. Reason: failed to account for genomic rearrangements, producing clusters lacking unique marker material
Advancing virome analysis: New computational methods for viral profiling and characterization with applications to inflammatory bowel disease · Harvard · PhD
Tried and failed
domain-level homology aggregation for annotation transfer applied to genetic variant pathogenicity classification. Outcome: worse than baseline. Reason: broader structural domain aggregation diluted signal compared to closer paralogous sequence alignments
Considered and rejected
Considered and rejected: Rejected using whole-protein sequence alignment for homology mining because variable repeat copy numbers and large sequence length discrepancies cause alignment failure and miss remote homologues.
Diversity and Evolution of Cyclic Peptides in Flax (Linum usitatissimum L.) · Saskatchewan · PhD
Considered and rejected
Considered and rejected: Aligning full-length multimodular NRPS sequences or whole modules for single phylogenetic tree construction was rejected due to large gaps and conflicting phylogenetic histories between A- and C-domains.
Drift, selection, and convergence in the evolution of a nonribosomal peptide · University of Marburg · PhD
Inadequate alignment depth and sequence power limit covariation and secondary structure prediction
Covariance analysis and thermodynamic structure predictors fail to identify functional RNA base pairs when sequence alignments lack sufficient evolutionary depth and diversity. Minimum free energy scores show weak correlation with empirical fitness, while predictors evaluated without evolutionary profiles fall below simple sequence-trained baselines.
Tried and failed
sequence covariation analysis on predicted thermodynamic structures applied to viral RNA secondary structure motifs. Outcome: no signal. Reason: failed to detect statistically significant covarying base pairs despite alignments having sufficient statistical power
Uncovering the structure and function of RNAs using computational and experimental approaches · Iowa State · PhD
Lost to a baseline
ViennaRNA failed to predict target secondary structures (returning NA) for 5 out of 7 extant yeast tRNA sequences, whereas RNAstructure succeeded for all 7.
Evolution of Essential and Ancient Genes: Understanding and Modeling Fitness in Transfer RNAs · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Rejected discarding structural motifs that lack statistically significant covariation, as sequence alignments of recent viral/mammalian origins often lack evolutionary depth.
High-throughput characterization of RNA structural motifs · Iowa State · PhD
Tried and failed
phylogenetic conservation scoring from small multi-species alignments applied to identifying constrained genomic sites. Outcome: no signal. Reason: insufficient phylogenetic depth and taxa number yielded low statistical power to detect conserved positions
Differential Dynamics of Deleterious Variation Across Demographically Diverse Florida Scrub-Jay Populations · Cornell · PhD
Tried and failed
covariance analysis for conserved secondary structure detection applied to conserved RNA hairpin motifs. Outcome: no signal. Reason: insufficient phylogenetic sequence alignment power to detect statistically significant covarying base pairs
Deciphering the RNA secondary structure landscape of human genes · Iowa State · PhD
Tried and failed
minimum free energy secondary structure folding scores applied to predicting RNA fitness from sequence variants. Outcome: no signal. Reason: folding free energy lacks linear correlation with empirical fitness and misses non-stem functional loop mutation impacts
Evolution of Essential and Ancient Genes: Understanding and Modeling Fitness in Transfer RNAs · Georgia Tech · PhD
Lost to a baseline
Profile-trained secondary structure predictor evaluated directly on sequences without profiles achieved only 68.6% accuracy, falling below the sequence-trained baseline model (72.3%)
Predicting Protein Structural Annotations by Deep and Shallow Learning · University College Dublin · PhD
Prohibitive computational overhead restricts exhaustive multiple sequence alignment at scale
Traditional multiple sequence alignments, tree-building algorithms, and all-versus-all long-read alignments require excessive compute time on large datasets. Practitioners reject exhaustive pairwise alignments in favor of k-mer clustering, single-sequence embeddings, or alignment-free conditional probability methods.
Considered and rejected
Considered and rejected: Rejected aligning all family member sequences with ensemble alignment methods due to non-reviewed sequences, excessive compute time, and alignment gaps/noise.
COMBINING HUERISTIC APPROACHES WITH MACHINE LEARNING TO RECOMMEND POST TRANSLATIONAL MODIFICATIONS FOR STUDY · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Decided against performing full all-versus-all alignments for every long read, opting instead for RATTLE's two-step bitvector and longest-increasing-subsequence k-mer clustering to avoid prohibitive computational overhead.
Computational Analysis of the Transcriptome Using Long-Read RNA Sequencing · Johns Hopkins · PhD
Considered and rejected
Considered and rejected: Rejected Needleman-Wunsch pairwise alignments for all reads in AmpliCI due to high computational cost; adopted an alignment-free conditional probability strategy.
Model-based clustering methods for high-throughput sequencing data · Iowa State · PhD
Considered and rejected
Considered and rejected: Rejected using traditional multiple sequence alignments (MSAs) or whole-genome graph traversals for large pangenome visualization due to prohibitive computational costs and failure under high sequence divergence.
Methods and applications for large-scale pangenomic analysis · Johns Hopkins · PhD
Considered and rejected
Considered and rejected: Rejected explicit tree construction (e.g., UPGMA or Neighbor-Joining) for progressive sequence alignment due to high computational burden when aligning many sequences, adopting iterative distance-matrix merging instead.
Developing a Multiday Travel Demand Modelling System · Dalhousie · Master's
Considered and rejected
Considered and rejected: Rejected multiple sequence alignments (MSAs) in favor of single-sequence PLM embeddings to eliminate high alignment computational overhead
Predicting One-Dimensional Protein Structures by Leveraging Pre-Trained Language Models (PLMs) and Deep Learning · University College Dublin · PhD
Alignment trimming and gap filtering remove informative phylogenetic sites or provide no benefit
Automated alignment trimming methods often exclude well-aligned and phylogenetically informative sites from sequence datasets. Furthermore, filtering gap positions or trimming concatenated whole-genome alignments frequently yields identical tree topologies without improving divergence time estimates.
Tried and failed
automated multiple sequence alignment trimming applied to divergent protein sequence datasets. Reason: consistently excluded blocks of well-aligned, informative phylogenetic sites
The evolution and diversity of noncanonical microbial nitrogen metabolisms · MIT · PhD
Tried and failed
alignment trimming prior to phylogenetic inference applied to concatenated maximum-likelihood phylogenomics. Outcome: no signal. Reason: trimmed alignments produced identical phylogenetic tree topologies to untrimmed alignments
The evolution of anthraquinones as an adaptive trait in lichen-forming fungi · Imperial · PhD
Tried and failed
filtering gap-rich columns in sequence alignments applied to phylogenetic divergence time estimation. Outcome: no signal. Reason: removing gap positions yielded no significant improvement in divergence time estimates
Bayesian methods for source attribution using HIV deep sequence data · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected using trimmed alignments for concatenated whole-genome phylogenetics after finding untrimmed alignments produced identical topologies.
The evolution of anthraquinones as an adaptive trait in lichen-forming fungi · Imperial · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Investigate mathematical and sequence correlations between the HIV-1 genome and the human host genome. Blocker: Lack of specific mathematical formulation, target human genomic regions, or methodology definition
Mathematical patterns associated with genetic recombination in HIV-1 and SARS-CoV-2 via explainability in CNNs: relationship of interpretability scoremaps with viral phylogeny and · University of Deusto · PhD
Left open
Develop computational models to map within-host viral evolutionary dynamics and spatial dissemination across tissue compartments using multi-tissue sequence data. Blocker: None
Examining viral pathogen evolution and spread through genomic data · Harvard · PhD
Left open
Develop and test a convolutional neural network that takes sequence alignment matrices as input to predict outbreak characteristics such as infection timing. Blocker: None
Evolution and population dynamics of mycobacterial pathogens with applications for disease control · Cornell · PhD
Left open
Test whether HSV-1 viral genomes exhibiting differential chromatin accessibility are differentially packaged into viral capsids. Blocker: Requires wet-lab experimental biology techniques to isolate capsids and sequence encapsidated viral genomes.
UNDERSTANDING THE ROLE OF HISTONE H2A VARIANTS IN THE GENOME-WIDE REGULATION OF HSV-1 LYTIC CHROMATIN DYNAMICS · Cornell · PhD
Left open
Basecall plasmid Nanopore Fast5 files using methylation-aware and standard modes, then compare error profiles against reference sequences. Blocker: Requires the raw Nanopore Fast5 sequencing data generated from the specific plasmid templates in the thesis.
ASSESING amplified aene aemplate quality with Nanopore sequencing for improved cell free expression · Iowa State · Master's
Left open
Test whether ST258 OmpK36 porin mutations confer similar carbapenem resistance phenotypes when introduced into Klebsiella pneumoniae sequence type ST307. Blocker: Requires a wet microbiology lab and genetic engineering protocols to introduce mutations into K. pneumoniae ST307 strains and measure resistance.
OmpK36-mediated carbapenem resistance in Klebsiella pneumoniae · Imperial · PhD
Left open
Analyze resistance genes in genome assemblies to identify predominant resistance mechanisms in poorly characterized Pseudomonas species. Blocker: Requires unpublished isolate sequence data or specific isolate strains generated in the thesis
Non-therapeutic antimicrobial resistance in wastewater and wastewater impacted surface waters: patterns, determinants and new approaches · Johns Hopkins · PhD
Left open
Analyze RNA sequences to discover sequence motifs or features associated with transcript colocalization patterns. Blocker: No specific hypothesis, sequence modeling method, or concrete target dataset provided beyond a broad research direction
Decoding Spatial Transcriptomics: Computational Frameworks for Subcellular Spatial Patterns and Contact Mediated Signaling · Georgia Tech · PhD
Left open
Infer macroinvertebrate phylogeny using combined nucleotide and amino acid sequence alignments. Blocker: None
Characterizing freshwater macroinvertebrates of Bangladesh using metagenetic techniques · Imperial · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Ling Wang · Georgia Tech · PhD · 2025
Evolution of Essential and Ancient Genes: Understanding and Modeling Fitness in Transfer RNAs - Theo Llewellyn · Imperial · PhD · 2023
The evolution of anthraquinones as an adaptive trait in lichen-forming fungi - Thomas Carter · Cornell · PhD · 2021
RECOMBINATION-MEDIATED REGULATORY EVOLUTION OF HUMAN ENDOGENOUS RETROVIRUS HERVH - Jordan S. L. Jensen · Harvard · PhD · 2026
Advancing virome analysis: New computational methods for viral profiling and characterization with applications to inflammatory bowel disease - Jaclyn Mary Long Walsh · Harvard · PhD · 2026
Single-Cell Biology of Respiratory Viral Infections in the Nasal Mucosa - Tal D Scully · Harvard · PhD · 2026
Evolution of Cell Types in Tunicate Blood - Abdul-Rahman Adamu Bukari · University of Manitoba · PhD · 2026
Genetic diversity of Candida albicans and Nakaseomyces glabratus across geographic scales and within-host populations - Blessy Antony · Virginia Tech · Doctorate · 2026
Discovering Viral Hosts, Mutations, and Diseases using Machine Learning - Anurendra Kumar · Georgia Tech · PhD · 2025
Decoding Spatial Transcriptomics: Computational Frameworks for Subcellular Spatial Patterns and Contact Mediated Signaling - Mitchell Lokey · Cornell · PhD · 2025
Differential Dynamics of Deleterious Variation Across Demographically Diverse Florida Scrub-Jay Populations - Brandon Roy · Cornell · PhD · 2025
THE HOST RESPONSE TO GRAPEVINE FANLEAF VIRUS AND IMPLICATIONS FOR SYMPTOMATOLOGY AND TRANSMISSION - Mengjun Xu · Harvard · Master's · 2025
Bioinformatic Sequence Analysis Reveals Evolutionary History of Synaptic Genes
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