diffcyt: Differential discovery in high-dimensional cytometry via high-resolution clustering

Weber, Lukas M.; Nowicka, Małgorzata; Soneson, Charlotte; Robinson, Mark D.

doi:10.1038/s42003-019-0415-5

Cited by 174 publications

(138 citation statements)

References 44 publications

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“…Neutrophils were in silico gated out and then re‐clustered. Changes in subpopulations abundance and differential states of the subpopulations were tested using the Diffcyt package …”

Section: Methodsmentioning

confidence: 99%

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis

Shaul¹,

Eyal²,

Guglietta

et al. 2020

FASEB j.

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The accumulation of circulating low‐density neutrophils (LDN) has been described in cancer patients and associated with tumor‐supportive properties, as opposed to the high‐density neutrophils (HDN). Here we aimed to evaluate the clinical significance of circulating LDN in lung cancer patients, and further assessed its diagnostic vs prognostic value. Using mass cytometry (CyTOF), we identified major subpopulations within the circulating LDN/HDN subsets and determined phenotypic modulations of these subsets along tumor progression. LDN were highly enriched in the low‐density (LD) fraction of advanced lung cancer patients (median 7.0%; range 0.2%‐80%, n = 64), but not in early stage patients (0.7%; 0.05%‐6%; n = 35), healthy individuals (0.8%; 0%‐3.5%; n = 15), or stable chronic obstructive pulmonary disease (COPD) patients (1.2%; 0.3%‐7.4%, n = 13). Elevated LDN (>10%) remarkably related with poorer prognosis in late stage patients. We identified three main neutrophil subsets which proportions are markedly modified in cancer patients, with CD66b+/CD10low/CXCR4+/PDL1inter subset almost exclusively found in advanced lung cancer patients. We found substantial variability in subsets between patients, and demonstrated that HDN and LDN retain a degree of inherent spontaneous plasticity. Deep phenotypic characterization of cancer‐related circulating neutrophils and their modulation along tumor progression is an important advancement in understanding the role of myeloid cells in lung cancer.

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“…Neutrophils were in silico gated out and then re‐clustered. Changes in subpopulations abundance and differential states of the subpopulations were tested using the Diffcyt package …”

Section: Methodsmentioning

confidence: 99%

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis

Shaul¹,

Eyal²,

Guglietta

et al. 2020

FASEB j.

Self Cite

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“…Future developments in this field will likely borrow from the mass cytometry (e.g. Tibshirani et al, 2002;Arvaniti & Claassen, 2017;Lun et al, 2017;Weber et al, 2018) or the microbiome literature (Gloor et al, 2017), where compositional data analysis has received more attention.…”

Section: Compositional Analysismentioning

confidence: 99%

Current best practices in single‐cell RNA‐seq analysis: a tutorial

Luecken

Theis

2019

Molecular Systems Biology

1,525

1,386

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Single‐cell RNA ‐seq has enabled gene expression to be studied at an unprecedented resolution. The promise of this technology is attracting a growing user base for single‐cell analysis methods. As more analysis tools are becoming available, it is becoming increasingly difficult to navigate this landscape and produce an up‐to‐date workflow to analyse one's data. Here, we detail the steps of a typical single‐cell RNA ‐seq analysis, including pre‐processing (quality control, normalization, data correction, feature selection, and dimensionality reduction) and cell‐ and gene‐level downstream analysis. We formulate current best‐practice recommendations for these steps based on independent comparison studies. We have integrated these best‐practice recommendations into a workflow, which we apply to a public dataset to further illustrate how these steps work in practice. Our documented case study can be found at https://www.github.com/theislab/single-cell-tutorial . This review will serve as a workflow tutorial for new entrants into the field, and help established users update their analysis pipelines.

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“…This model takes into account the distribution of each marker, and has a donor-specific variable to control for inter-individual variability. For the clustering analyses, the R package CATALYST was used (Nowicka et al, 2017;Weber et al, 2019). This package provides a clustering method which combines the FlowSOM algorithm (Van Gassen et al, 2015) which generates 100 high-resolution clusters, followed by the ConsensusClusterPlus metaclustering algorithm (Wilkerson and Hayes, 2010) which regroups these high-resolution clusters into metaclusters.…”

Section: Discussionmentioning

confidence: 99%

“…Default parameters were used for clustering, and the number of metaclusters (10) was selected based on the delta area plot provided. To test for differential abundance of clusters between groups, the diffcyt-DA-GLMM method from the diffcyt package (Nowicka et al, 2017;Weber et al, 2019) was used; the donor IDs were specified as a random effect. The UMAP was run using the scater package (McCarthy et al, 2017), with default settings.…”

Section: Discussionmentioning

confidence: 99%

Treated HIV Infection Alters Phenotype But Not HIV-specific Function of Peripheral Blood Natural Killer Cells

Zhao

Ferreira

Grant

et al. 2020

Preprint

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ABSTRACTNatural killer (NK) cells are the predominant antiviral cells of the innate immune system, and may play an important role in acquisition and disease progression of HIV. While untreated HIV infection is associated with distinct alterations in the peripheral blood NK cell repertoire, less is known about how NK phenotype is altered in the setting of long-term viral suppression with antiretroviral therapy (ART), as well as how NK memory can impact functional responses. As such, we sought to identify changes in NK cell phenotype and function using high-dimensional mass cytometry to simultaneously analyze both surface and functional marker expression of peripheral blood NK cells in a cohort of ART-suppressed, HIV+ patients and HIV-healthy controls. We found that the NK cell repertoire following IL-2 treatment was altered in individuals with treated HIV infection compared to healthy controls, with increased expression of markers including NKG2C and CD2, and decreased expression of CD244 and NKp30. Using co-culture assays with autologous, in vitro HIV-infected CD4 T cells, we identified a subset of NK cells with enhanced responsiveness to HIV-1-infected cells, but no differences in the magnitude of anti-HIV NK cell responses between the HIV+ and HIV-groups. In addition, by profiling of NK cell receptors on responding cells, we found similar phenotypes of HIV-responsive NK cell subsets in both groups. Lastly, we identified clusters of NK cells that are altered in individuals with treated HIV infection compared to healthy controls, but found that these clusters are distinct from those that respond to HIV in vitro. As such, we conclude that while chronic, treated HIV infection induces a reshaping of the IL-2-stimulated peripheral blood NK cell repertoire, it does so in a way that does not make the repertoire more HIV-specific.

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diffcyt: Differential discovery in high-dimensional cytometry via high-resolution clustering

Cited by 174 publications

References 44 publications

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis

Current best practices in single‐cell RNA‐seq analysis: a tutorial

Treated HIV Infection Alters Phenotype But Not HIV-specific Function of Peripheral Blood Natural Killer Cells

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