Alexa Noelle Gormick

All Publications

• ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples. Nucleic acids research Zahm, A. M., Cranney, C. W., Gormick, A. N., Rondem, K. E., Schmitz, B., Himes, S. R., English, J. G. 2025; 53 (22)

  Abstract

  Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform's performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of severe acute respiratory syndrome coronavirus 2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research.

  View details for DOI 10.1093/nar/gkaf1304

  View details for PubMedID 41385323

  View details for PubMedCentralID PMC12700093

• High-Throughput Characterization of Tetracycline Repressor Function on Tetracycline Operator 2 Variants. ACS synthetic biology Gormick, A. N., Zahm, A. M., Himes, S. R., Rondem, K. E., English, J. G. 2025; 14 (6): 1912-1919

  Abstract

  Chemogenetic regulators of transgene activity, such as the tetracycline-inducible system derived from the tetracycline resistance operon of the bacterial transposon Tn10, are critical and widely used systems in cellular engineering. The tetracycline-inducible system is prized for its selectivity, high affinity, inducibility, reversibility, and differential control of gene transcription. However, its optimization for binary on/off expression limits its application in systems biology and the modeling and construction of complex regulatory systems with intricate input/output paradigms. To overcome this limitation, we developed a high-throughput reporter system to investigate a saturated mutagenesis library of tetracycline resistance operator variants. Using this system, we mapped the functional interactions of Tet repressor DNA binding protein at single-nucleotide resolution in mammalian cells. Our comprehensive screen revealed a spectrum of variant effects, ranging from a nearly complete loss of repression to levels indistinguishable from the natural operator, validated through orthogonal assays. This comprehensive characterization of the sequence-specificity of a tetracycline resistance operator facilitates the construction of variably suppressive, inducible systems for dynamic and modular control over gene expression in mammalian cell culture.

  View details for DOI 10.1021/acssynbio.4c00809

  View details for PubMedID 40489706

• A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells. Nature communications Zahm, A. M., Owens, W. S., Himes, S. R., Fallon, B. S., Rondem, K. E., Gormick, A. N., Bloom, J. S., Kosuri, S., Chan, H., English, J. G. 2024; 15 (1): 10353

  Abstract

  Cellular responses to stimuli underpin discoveries in drug development, synthetic biology, and general life sciences. We introduce a library comprising 6144 synthetic promoters, each shorter than 250 bp, designed as transcriptional readouts of cellular stimulus responses in massively parallel reporter assay format. This library facilitates precise detection and amplification of transcriptional activity from our promoters, enabling the systematic development of tunable reporters with dynamic ranges of 50-100 fold. Our library proved functional in numerous cell lines and responsive to a variety of stimuli, including metabolites, mitogens, toxins, and pharmaceutical agents, generating robust and scalable reporters effective in screening assays, biomarkers, and synthetic circuits attuned to endogenous cellular activities. Particularly valuable in therapeutic development, our library excels in capturing candidate reporters to signals mediated by drug targets, a feature we illustrate across nine diverse G-protein coupled receptors (GPCRs), critical targets in drug development. We detail how this tool isolates and defines discrete signaling pathways associated with specific GPCRs, elucidating their transcriptional signatures. With its ease of implementation, broad utility, publicly available data, and comprehensive documentation, our library will be beneficial in synthetic biology, cellular engineering, ligand exploration, and drug development.

  View details for DOI 10.1038/s41467-024-54502-9

  View details for PubMedID 39609378

  View details for PubMedCentralID PMC11604768