Biology subjects

Levi, E.

Publications and source records attributed to Levi, E..

3 recordsLinked to original sources

bioRxiv2026

Order Matters: Ambient RNA correction confounds doublet detection in single-cell RNA sequencing

A common technical artifact in droplet-based single-cell RNA sequencing (scRNA-seq) is the capture of two cells in the same droplet, termed doublets. Doublets may present as hybrid cell types, and their removal is a critical step during quality control. Another form of contamination is ambient RNA, which originates from free floating RNA released from other cells in suspension, likely during cell death. As with doublets, ambient RNA results in unexpected gene expression patterns. Most suggested analysis pipelines include ambient RNA correction before doublet detection however no formal investigation into how doublet detection is affected by ambient RNA correction has been conducted. Here, we evaluate the impact of ambient RNA correction on the performance of doublet detection using novel engineered and multiplexed datasets. We apply three of the most widely-used methods for removing ambient RNA (decontX, soupX, CellBender), in conjunction with up to six doublet detection methods (scDblFinder, scds, bcds, cxds, scrublet, DoubletDetection). We find that doublet detection tools perform best when done prior to ambient RNA correction, with the exception of CellBender. Performing ambient RNA correction first, particularly for decontX, leads to an increased proportion of doublet droplets retained in the dataset compared with calling doublets on uncorrected data. We show that both decontX and soupX partially mask doublet signal by removing proportionally more ambient RNA from doublets that remain undetected after ambient RNA correction. Our results show that the performance of doublet detection is significantly impacted by the use of ambient RNA correction tools. We suggest a new workflow that implements doublet detection prior to ambient RNA correction unless using CellBender.

bioinformatics↗

bioRxiv2026

Single-cell CRISPR activation screens in primary B cells discover gene regulatory mechanisms for hundreds of autoimmune risk loci.

Genome-wide association studies (GWAS) have discovered thousands of genetic variants linked to autoimmune disease, and yet the molecular pathways underlying autoimmunity have remained elusive. A key challenge is that >90% of identified GWAS risk loci are in non-coding genomic regions making it difficult to predict their relevance to disease. Here, we have curated fine-mapped non-coding risk variants from over 30 different autoimmune traits including common conditions such as systemic lupus erythematosus (SLE), Crohns disease, and multiple sclerosis, and reveal shared genetic signatures between diverse autoimmune diseases. We subsequently performed a high-throughput single-cell multi-omic CRISPR activation screen targeting 763 autoimmune risk loci in primary human B cells (a highly relevant cell type to autoimmune diseases) and discover 524 cis-regulatory target gene effects for 378 risk loci, with many risk loci regulating multiple gene targets. This Single Cell Analysis of Non-coding Distal Autoimmune Loci (SCANDAL) provides a powerful experimental resource linking non-coding risk loci to many disease-relevant genes, including lowly-expressed cytokines and transcription factors for which perturbation effects can be difficult to quantify with other CRISPR-based strategies. We reveal how increased transcriptional activity at one non-coding risk locus can drive transcription at other risk loci within the same regulatory landscape that may be relevant to understand genetic pleiotropy of autoimmune diseases. Finally, we quantified allele-specific effects on target gene expression with massive parallel reporter assays and prime editing to discover a gain-of-function variant associated with SLE that controls expression of the transcription factor REL/cREL which subsequently binds dozens of risk loci and target genes associated with different autoimmune diseases. Our study provides a valuable resource linking non-coding risk loci with their cis-regulatory target genes and advances our understanding of the shared genetic networks and mechanisms involved in autoimmunity.

genomics↗

bioRxiv2025

Antibody responses against bacterial glycans affinity mature and diversify in germinal centers.

Anti-carbohydrate antibodies (Abs) play crucial roles in pathogen control, but their generation remains poorly understood. By studying responses to Streptococcus pyogenes in humans, we reveal that the glycan-targeted response shifts from IgM towards IgG and IgA memory with age and antigen exposure across blood, spleen, and tonsils. Both natural colonization and controlled human infection with S. pyogenes increased class-switched B cells, with evidence of within-clone switching. Glycan-specific B cells readily participated in germinal center (GC) responses and showed robust somatic hypermutation despite a molecular signature consistent with receiving reduced T cell help. We conclude that mucosal pathogen encounters elicit glycan responses that class-switch, evolve and diversify through the GC. These findings reveal how age and infection history can influence the quality, quantity, and isotype use of glycan-specific B cells, with implications for the design and schedule of glycan-containing vaccines.

immunology↗