Frontiers in Immunology (Aug 2025)
An immune-focused supplemental alignment pipeline captures information missed from dominant single-cell RNA-seq analyses, including allele-specific MHC-I regulation
- Sebastian Benjamin,
- GW McElfresh,
- Maanasa Kaza,
- Gregory J. Boggy,
- Benjamin Varco-Merth,
- Benjamin Varco-Merth,
- Sohita Ojha,
- Shana Feltham,
- William Goodwin,
- William Goodwin,
- Candice Nkoy,
- Candice Nkoy,
- Derick Duell,
- Derick Duell,
- Andrea Selseth,
- Tyler Bennett,
- Aaron Barber-Axthelm,
- Nicole N. Haese,
- Nicole N. Haese,
- Helen Wu,
- Courtney Waytashek,
- Carla Boyle,
- Jeremy V. Smedley,
- Jeremy V. Smedley,
- Caralyn S. Labriola,
- Caralyn S. Labriola,
- Michael K. Axthelm,
- Michael K. Axthelm,
- R. Keith Reeves,
- R. Keith Reeves,
- Daniel N. Streblow,
- Daniel N. Streblow,
- Jonah B. Sacha,
- Jonah B. Sacha,
- Afam A. Okoye,
- Afam A. Okoye,
- Scott G. Hansen,
- Scott G. Hansen,
- Louis J. Picker,
- Louis J. Picker,
- Benjamin N. Bimber,
- Benjamin N. Bimber
Affiliations
- Sebastian Benjamin
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- GW McElfresh
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Maanasa Kaza
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Gregory J. Boggy
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Benjamin Varco-Merth
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Benjamin Varco-Merth
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Sohita Ojha
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Shana Feltham
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- William Goodwin
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- William Goodwin
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Candice Nkoy
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Candice Nkoy
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Derick Duell
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Derick Duell
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Andrea Selseth
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Tyler Bennett
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Aaron Barber-Axthelm
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Nicole N. Haese
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Nicole N. Haese
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Helen Wu
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Courtney Waytashek
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Carla Boyle
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Jeremy V. Smedley
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Jeremy V. Smedley
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Caralyn S. Labriola
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Caralyn S. Labriola
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Michael K. Axthelm
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Michael K. Axthelm
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- R. Keith Reeves
- Division of Innate and Comparative Immunology, Center for Human Systems Immunology, Duke University School of Medicine, Durham, NC, United States
- R. Keith Reeves
- Department of Surgery, Duke University School of Medicine, Durham, NC, United States
- Daniel N. Streblow
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Daniel N. Streblow
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Jonah B. Sacha
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Jonah B. Sacha
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Afam A. Okoye
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Afam A. Okoye
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Scott G. Hansen
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Scott G. Hansen
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Louis J. Picker
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Louis J. Picker
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- Benjamin N. Bimber
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, United States
- Benjamin N. Bimber
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR, United States
- DOI
- https://doi.org/10.3389/fimmu.2025.1596760
- Journal volume & issue
-
Vol. 16
Abstract
IntroductionRNA sequencing (RNA-seq) can measure whole transcriptome gene expression from tissues or even individual cells, providing a powerful tool to study the immune response. Analysis of RNA-seq data involves mapping relatively short sequence reads to a reference genome, and quantifying genes based on the position of alignments relative to annotated genes. While this is usually robust, genetic polymorphism or genome/annotation inaccuracies result in genes with systematically missing or inaccurate data. These issues are frequently hidden or ignored, yet are highly relevant to immunologic data, where balancing selection has generated many polygenic gene families not accurately represented in a ‘one-size-fits-all’ reference genome.MethodsHere we present nimble, a tool to supplement standard RNA-seq pipelines. Nimble uses a previously developed pseudoaligner to process either bulk- or single-cell RNA-seq data using custom gene spaces. Importantly, nimble can apply customizable scoring criteria to each gene set, tailored to the biology of those genes.ResultsWe demonstrate that nimble recovers data in diverse contexts, ranging from simple cases (e.g., incorrect gene annotation or viral RNA), to complex immune genotyping (e.g., major histocompatibility or killer-immunoglobulin-like receptors). We use this enhanced capability to identify killer-immunoglobulin-like receptor expression specific to tissue-resident memory T cells and demonstrate allele-specific regulation of MHC alleles after Mycobacterium tuberculosis stimulation.DiscussionCombining nimble data with standard pipelines enhances the fidelity and accuracy of experiments, maximizing the value of expensive datasets, and identifying cellular subsets not possible with standard tools alone.
Keywords
- single-cell RNA-seq (scRNA-seq)
- T cells
- bioinformatics
- immunogenetics
- major histocompatability complex (MHC)
