Double-jeopardy: scRNA-seq doublet/multiplet detection using multi-omic profiling

Sun, Bo; Bugarin-Estrada, Emmanuel; Overend, Lauren; Walker, Catherine Elizabeth; Tucci, Felicia Anna; Bashford-Rogers, Rachael

doi:10.1016/j.crmeth.2021.100008

Cited by 15 publications

(17 citation statements)

References 21 publications

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“…Doublet identification and removal was performed using both DoubletFinder 67 and MLtiplet 68 . Each cell type was subsetted into individual objects, and re-clustering within these objects was performed excluding genes which were likely to be influenced by experimental rather than biological factors 69 .…”

Section: Methodsmentioning

confidence: 99%

“…These clusters were then annotated broadly into B cell, T cell or myeloid clusters based on mapping of >10% BCR+ droplets and elevated CD19 expression, >10% TCR+ droplets and elevated CD3 expression, <10% BCR/TCR+ droplets, respectively. Doublet identification and removal was performed using both DoubletFinder 67 and MLtiplet 68 . Each cell type was subsetted into individual objects, and re-clustering within these objects was performed excluding genes which were likely to be influenced by experimental rather than biological factors 69 .…”

Section: Filtering Doublet Detection and Batch Correction Of The Panc...mentioning

confidence: 99%

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Single-cell immune multi-omics and repertoire analyses in pancreatic ductal adenocarcinoma reveal differential immunosuppressive mechanisms within different tumour microenvironments

Sivakumar,

Jainarayanan,

Arbe-Barnes

et al. 2023

Preprint

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Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. Understanding the multiple mechanisms by which the tumour evades immune control, and how these mechanisms may be disrupted is critical to developing targeted immunotherapies. Previous studies have shown that higher lymphocyte infiltration is associated with better survival, and here we investigated what mediates these differences. We performed a comprehensive analysis of PDAC-associated immune cells using single cell multi-omics coupled with re-analysis of public PDAC scRNA-seq datasets. We introduce novel single-cell and repertoire analyses that have uncoupled diverse roles and contributions of various immune cell populations within different tumour microenvironments (TMEs). They revealed clear distinctions in the clonal characteristics among different patient groups, provided valuable insights into the mechanisms of immune cell migration and tissue adaptation underlying these disparities. These results point to differential CD4 polarisation of intra-tumoural T cells, differential B cell differentiation, GC reactions, antigen presentation pathways, and distinct cell-cell communication between the myeloid-enriched and adaptive-enriched groups. Overall, we identified two major distinct themes for future immune intervention within PDAC patients between those with higher adaptive versus myeloid immune cell infiltration.

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Section: Methodsmentioning

confidence: 99%

Section: Filtering Doublet Detection and Batch Correction Of The Panc...mentioning

confidence: 99%

Single-cell immune multi-omics and repertoire analyses in pancreatic ductal adenocarcinoma reveal differential immunosuppressive mechanisms within different tumour microenvironments

Sivakumar,

Jainarayanan,

Arbe-Barnes

et al. 2023

Preprint

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“…The remaining 5% of nuclei (randomly distributed across the males) had alleles from both lineages, a result that most likely arose because of cross-contamination between different samples. A certain degree of such contamination is expected because it is challenging to accurately sort and genotype individual nuclei ( 12 , 13 ). To corroborate these results, we used whole-genome sequencing to identify R- and W-specific sequences and carried out in situ hybridization using probes targeting these sequences on male tissue sections.…”

Section: Males Are Chimeras Of Haploid Cells From the Two Divergent L...mentioning

confidence: 99%

Obligate chimerism in male yellow crazy ants

Darras

Berney

Hasin

et al. 2023

Science

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Multicellular organisms typically develop from a single fertilized egg and therefore consist of clonal cells. We report an extraordinary reproductive system in the yellow crazy ant. Males are chimeras of haploid cells from two divergent lineages: R and W. R cells are overrepresented in the males’ somatic tissues, whereas W cells are overrepresented in their sperm. Chimerism occurs when parental nuclei bypass syngamy and divide separately within the same egg. When syngamy takes place, the diploid offspring either develops into a queen when the oocyte is fertilized by an R sperm or into a worker when fertilized by a W sperm. This study reveals a mode of reproduction that may be associated with a conflict between lineages to preferentially enter the germ line.

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“…The hashtag negatives or doublets, as well as cells with low (<500) unique molecular identifiers (UMIs), were removed. Cells with <100 genes and >50% mitochondrial content were removed to filter low-quality, dead, or dying populations, with a second pass filter excluding apoptotic populations characterized by a low percentage of ribosomal genes versus a high percentage of mitochondrial genes, using a defined mitochondrial-ribosomal RNA ratio of >0.47 26 . Downstream transcriptome-based graph clustering and principal component analyses (PCA) with cell phenotype consensus calling were performed using R Seurat v4.1.1 package 9 , without input of TCR, BCR, mitochondrial, ribosomal, or sex-linked genes to avoid bias due to known expression variability.…”

Section: Single-cell 5' Rna-tcr-cite-sequencingmentioning

confidence: 99%

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

Gibson,

Ram,

Gangula

et al. 2023

Preprint

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Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but lifethreatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T-cells. To obtain an unbiased assessment of SJS/TEN cellular immunopathogenesis, we performed single-cell (sc) transcriptome, surface proteome, and TCR sequencing on unaffected skin, affected skin, and blister fluid from 17 SJS/TEN patients. From 119,784 total cells, we identified 16 scRNA-defined subsets, confirmed by subsetdefining surface protein expression. Keratinocytes upregulated HLA and IFN-response genes in the affected skin. Cytotoxic CD8+ T-cell subpopulations of expanded and unexpanded TCRαβ clonotypes were shared in affected skin and blister fluid but absent or unexpanded in SJS/TEN unaffected skin. SJS/TEN blister fluid is a rich reservoir of oligoclonal CD8+ T-cells with an effector phenotype driving SJS/TEN pathogenesis. This multiomic database will act as the basis to define antigen-reactivity, HLA restriction, and signatures of drug-antigen-reactive T-cell clonotypes at a tissue level.

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Double-jeopardy: scRNA-seq doublet/multiplet detection using multi-omic profiling

Cited by 15 publications

References 21 publications

Single-cell immune multi-omics and repertoire analyses in pancreatic ductal adenocarcinoma reveal differential immunosuppressive mechanisms within different tumour microenvironments

Single-cell immune multi-omics and repertoire analyses in pancreatic ductal adenocarcinoma reveal differential immunosuppressive mechanisms within different tumour microenvironments

Obligate chimerism in male yellow crazy ants

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

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