Biology subjects

Krebs, Y.

Publications and source records attributed to Krebs, Y..

3 recordsLinked to original sources

bioRxiv2026

Sample barcoding-associated technical variation in probe-based single-cell RNA sequencing

Probe-based single cell RNA sequencing approaches are increasingly becoming a technology of choice for profiling gene expression at scale and in archival tissues. The 10x Genomics Flex v1 assay enables cost-effective and high-sensitivity single-cell RNA sequencing by splitting samples across up to 16 uniquely barcoded probe sets before pooling and loading onto a single lane of a microfluidic chip. A natural consequence of this design is to leverage probe set barcoding as a sample barcoding strategy for case-control experiments. However, we observed that Flex v1 probe set barcode identity drives substantial technical variation between probe set barcodes, an effect that is reproducible across lanes and independent datasets. When Flex v1 probe set barcodes are confounded with biological sample identity, a concerning number of differentially expressed genes at standard thresholds are false positives. The Flex v2 assay, which decouples sample barcoding from probe set hybridization, significantly reduces this artifact. As the field continues to expand adoption of probe-based assays, our findings introduce probe set barcoding as an underappreciated source of technical variation in single-cell assays and emphasize the importance of experimental design when using probe-based sequencing technologies.

genomics↗

bioRxiv2024

TSniffer: Unbiased de novo identification of RNA editing sites and quantification of editing activity in RNA-seq data

RNA editing by adenosine deaminases acting on RNA (ADARs) is an evolutionarily conserved posttranscriptional modification essential for organismal development and normal cell function. Three catalytically active ADARs are conserved in mammals: two isoforms of ADAR1 referred to as ADAR1-p150 and ADAR1-p110, as well as ADAR2. All recognize and edit double-stranded RNA (dsRNA) structures but demonstrate target specificity and selectivity that dictate the unique essential biological functions of the three enzymes. The editing activity of ADAR1-p150 suppresses autoimmune responses against self-RNA structures, whereas ADAR1-p110 and ADAR2 have other primary functions. To better understand the mechanism of target selection by ADARs, we developed TSniffer, which allows accurate de novo identification of edited transcripts and quantification of the extent of editing within each transcript. We found that 17-40% of protein coding transcripts in mice, ferrets, and humans are edited by ADARs. Individual transcripts can harbor hundreds and thousands of editing sites, mostly within inverted retrotransposable elements. For human transcripts, we found differential editing by ADAR1 and ADAR2, aligning with a supportive role for ADAR2, while some targets were dominantly edited by ADAR1. Relying only on RNA-seq data and reference genome, TSniffer represents a novel tool to decipher the role of ADAR editing in different physiological states including disease models. Its unbiased approach is suitable for any organism.

bioinformatics↗

bioRxiv2022

Non-neutralizing antibody responses to vesicular stomatitis virus-vectored influenza A virus vaccines correlate with protection

Seasonal influenza virus infections remain a major global public health burden. In addition, influenza A virus (IAV) exhibits high pandemic potential through zoonotic spread from avian hosts to humans. Currently licensed IAV vaccines are mainly directed against the immuno-dominant surface protein hemagglutinin (HA). Since HA is antigenically highly variable, IAV can escape vaccine-derived immunity through antigenic drift. However, vaccine preparations such as the live-attenuated influenza vaccine (LAIV) also contain other more conserved viral antigens, whose contribution to influenza immunity are not fully elucidated. To determine the extent to which conserved LAIV antigens contribute to establishing protective immunity against heterologous IAV strains, we generated vesicular stomatitis virus-based single-round vector vaccines expressing individual LAIV antigens, and tested their ability to protect mice from a heterologous challenge with two IAV strains, PR8[H1N1] and rSC35M[H7N7]. We found that immunization with nucleoprotein (NP), ion channel M2, and the stem-region of HA (HAstem), but not matrix protein (M1), provide protection from severe disease caused by either IAV strain. This effect correlated with development of non-neutralizing antibodies cross-reactive with both virus strains. Notably, the individual antigens induced specific IgG subclass profiles with different reactivity against PR8 and rSC35M. Sera from vaccinated animals activated Fc-gamma receptor IV-mediated effector functions, suggesting that they can induce cell-mediated immune defense mechanisms, such as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Combination of the most potent antigens NP and M2 in a mixed vaccination resulted in enhanced protection against IAV challenge, suggesting that the antibody responses against these antigens were synergistic. Our results demonstrate the potency of NP and M2 proteins to serve as conserved antigen targets, resulting in broad protection against severe IAV disease.

immunology↗