Category Ranking
98%
Total Visits
921
Avg Visit Duration
2 minutes
Citations
20
Article Abstract
Background: Single-cell RNA sequencing (scRNA-seq) has improved our ability to characterize rare cell populations. In practice, cells from different tissues or donors are simultaneously loaded onto the instrument (multiplexed) at the recommended (standard loading) or higher (superloading) numbers to save time and money. Although cost-effective, superloading can stymie computational analyses owing to high multiplet rates and sample complexity.
Methods: We compared the effects of superloading on multiplexed single-cell gene expression and T cell receptor (TCR) data generated from human thymus and blood samples from different donors.
Results: Minimal transcriptomic differences were observed between the data generated by either standard or superloading. Irrespective of the loading cell number, we found that over 50% of the T cells expressing multiple TCR chains were doublets.
Conclusion: Multiple samples can be run simultaneously without compromising data quality and subsequent analyses. However, an additional doublet removal step based on TCR configuration may improve the accuracy of T cell analysis.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1080/08820139.2025.2457039 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Benchmarking of computational demultiplexing methods for single-nucleus RNA sequencing data.
Brief Bioinform
July 2025
Laboratory of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Herestraat 49, 3000 Leuven, Belgium.
Single-nucleus RNA sequencing (snRNA-Seq) has transformed our understanding of complex tissues, providing insights into cellular composition and heterogeneity in gene expression between cells, and their alterations in development and disease. High costs however constrain the number of samples analysed. Sample pooling and their demultiplexing following sequencing based on prior labelling with antibodies or lipid anchors conjugated to DNA barcodes (cell hashing and MULTI-seq), or using genetic differences between samples, provides a solution.
View Article and Find Full Text PDFAlpha-tubulin tails regulate axoneme differentiation.
Proc Natl Acad Sci U S A
April 2025
Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
The tubulin tail is a key element for microtubule (MT) functionality, but the functional redundancy of tubulin genes complicates the genetic determination of their physiological functions. Here, we removed the C-terminal tail of five alpha- and four beta-tubulin genes in the genome. Sensory cilia typically exhibit an axoneme that longitudinally differentiates into a middle segment with doublet MTs and a distal segment with singlet MTs.
View Article and Find Full Text PDFReducing batch effects in single cell chromatin accessibility measurements by pooled transposition with MULTI-ATAC.
bioRxiv
February 2025
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
Large-scale scATAC-seq experiments are challenging because of their costs, lengthy protocols, and confounding batch effects. Several sample multiplexing technologies aim to address these challenges, but do not remove batch effects introduced when performing transposition reactions in parallel. We demonstrate that sample-to-sample variability in nuclei-to-Tn5 ratios is a major cause of batch effects and develop MULTI-ATAC, a multiplexing method that pools samples prior to transposition, as a solution.
View Article and Find Full Text PDFThe impact of ambient contamination on demultiplexing methods for single-nucleus multiome experiments.
Res Sq
February 2025
Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles.
Sample multiplexing has become an increasingly common design choice in droplet-based single-nucleus multi-omic sequencing experiments to reduce costs and remove technical variation. Genotype-based demultiplexing is one popular class of methods that was originally developed for single-cell RNA-seq, but has not been rigorously benchmarked in other assays, such as snATAC-seq and joint snRNA/snATAC assays, especially in the context of variable ambient RNA/DNA contamination. To address this, we develop ambisim, a genotype-aware read-level simulator that can flexibly control ambient molecule proportions and generate realistic joint snRNA/snATAC data.
View Article and Find Full Text PDFThe impact of ambient contamination on demultiplexing methods for single-nucleus multiome experiments.
bioRxiv
February 2025
Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles.
Sample multiplexing has become an increasingly common design choice in droplet-based single-nucleus multi-omic sequencing experiments to reduce costs and remove technical variation. Genotype-based demultiplexing is one popular class of methods that was originally developed for single-cell RNA-seq, but has not been rigorously benchmarked in other assays, such as snATAC-seq and joint snRNA/snATAC assays, especially in the context of variable ambient RNA/DNA contamination. To address this, we develop ambisim, a genotype-aware read-level simulator that can flexibly control ambient molecule proportions and generate realistic joint snRNA/snATAC data.
View Article and Find Full Text PDF