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Article ; Online: scruff

Zhe Wang / Junming Hu / W. Evan Johnson / Joshua D. Campbell

BMC Bioinformatics, Vol 20, Iss 1, Pp 1-

an R/Bioconductor package for preprocessing single-cell RNA-sequencing data

2019 Volume 9

Abstract: Abstract Background Single-cell RNA sequencing (scRNA-seq) enables the high-throughput quantification of transcriptional profiles in single cells. In contrast to bulk RNA-seq, additional preprocessing steps such as cell barcode identification or unique ... ...

Abstract	Abstract Background Single-cell RNA sequencing (scRNA-seq) enables the high-throughput quantification of transcriptional profiles in single cells. In contrast to bulk RNA-seq, additional preprocessing steps such as cell barcode identification or unique molecular identifier (UMI) deconvolution are necessary for preprocessing of data from single cell protocols. R packages that can easily preprocess data and rapidly visualize quality metrics and read alignments for individual cells across multiple samples or runs are still lacking. Results Here we present scruff, an R/Bioconductor package that preprocesses data generated from the CEL-Seq or CEL-Seq2 protocols and reports comprehensive data quality metrics and visualizations. scruff rapidly demultiplexes, aligns, and counts the reads mapped to genome features with deduplication of unique molecular identifier (UMI) tags. scruff also provides novel and extensive functions to visualize both pre- and post-alignment data quality metrics for cells from multiple experiments. Detailed read alignments with corresponding UMI information can be visualized at specific genome coordinates to display differences in isoform usage. The package also supports the visualization of quality metrics for sequence alignment files for multiple experiments generated by Cell Ranger from 10X Genomics. scruff is available as a free and open-source R/Bioconductor package. Conclusions scruff streamlines the preprocessing of scRNA-seq data in a few simple R commands. It performs data demultiplexing, alignment, counting, quality report and visualization systematically and comprehensively, ensuring reproducible and reliable analysis of scRNA-seq data.
Keywords	Scruff ; Single-cell RNA-sequencing ; Cell barcode demultiplexing ; Unique molecular identifier (UMI) ; Visualization of data quality ; Computer applications to medicine. Medical informatics ; R858-859.7 ; Biology (General) ; QH301-705.5
Subject code	004
Language	English
Publishing date	2019-05-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Bulk brain tissue cell-type deconvolution with bias correction for single-nuclei RNA sequencing data using DeTREM

Nicholas K. O’Neill / Thor D. Stein / Junming Hu / Habbiburr Rehman / Joshua D. Campbell / Masanao Yajima / Xiaoling Zhang / Lindsay A. Farrer

BMC Bioinformatics, Vol 24, Iss 1, Pp 1-

2023 Volume 16

Abstract: Abstract Background Quantifying cell-type abundance in bulk tissue RNA-sequencing enables researchers to better understand complex systems. Newer deconvolution methodologies, such as MuSiC, use cell-type signatures derived from single-cell RNA-sequencing ...

Abstract	Abstract Background Quantifying cell-type abundance in bulk tissue RNA-sequencing enables researchers to better understand complex systems. Newer deconvolution methodologies, such as MuSiC, use cell-type signatures derived from single-cell RNA-sequencing (scRNA-seq) data to make these calculations. Single-nuclei RNA-sequencing (snRNA-seq) reference data can be used instead of scRNA-seq data for tissues such as human brain where single-cell data are difficult to obtain, but accuracy suffers due to sequencing differences between the technologies. Results We propose a modification to MuSiC entitled ‘DeTREM’ which compensates for sequencing differences between the cell-type signature and bulk RNA-seq datasets in order to better predict cell-type fractions. We show DeTREM to be more accurate than MuSiC in simulated and real human brain bulk RNA-sequencing datasets with various cell-type abundance estimates. We also compare DeTREM to SCDC and CIBERSORTx, two recent deconvolution methods that use scRNA-seq cell-type signatures. We find that they perform well in simulated data but produce less accurate results than DeTREM when used to deconvolute human brain data. Conclusion DeTREM improves the deconvolution accuracy of MuSiC and outperforms other deconvolution methods when applied to snRNA-seq data. DeTREM enables accurate cell-type deconvolution in situations where scRNA-seq data are not available. This modification improves characterization cell-type specific effects in brain tissue and identification of cell-type abundance differences under various conditions.
Keywords	Deconvolution ; Single-nuclei RNA sequencing ; Brain cell-types ; MuSiC ; Computer applications to medicine. Medical informatics ; R858-859.7 ; Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2023-09-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A contamination focused approach for optimizing the single-cell RNA-seq experiment

Deronisha Arceneaux / Zhengyi Chen / Alan J. Simmons / Cody N. Heiser / Austin N. Southard-Smith / Michael J. Brenan / Yilin Yang / Bob Chen / Yanwen Xu / Eunyoung Choi / Joshua D. Campbell / Qi Liu / Ken S. Lau

iScience, Vol 26, Iss 7, Pp 107242- (2023)

2023

Abstract: Summary: Droplet-based single-cell RNA-seq (scRNA-seq) data are plagued by ambient contaminations caused by nucleic acid material released by dead and dying cells. This material is mixed into the buffer and is co-encapsulated with cells, leading to a ... ...

Abstract	Summary: Droplet-based single-cell RNA-seq (scRNA-seq) data are plagued by ambient contaminations caused by nucleic acid material released by dead and dying cells. This material is mixed into the buffer and is co-encapsulated with cells, leading to a lower signal-to-noise ratio. Although there exist computational methods to remove ambient contaminations post-hoc, the reliability of algorithms in generating high-quality data from low-quality sources remains uncertain. Here, we assess data quality before data filtering by a set of quantitative, contamination-based metrics that assess data quality more effectively than standard metrics. Through a series of controlled experiments, we report improvements that can minimize ambient contamination outside of tissue dissociation, via cell fixation, improved cell loading, microfluidic dilution, and nuclei versus cell preparation; many of these parameters are inaccessible on commercial platforms. We provide end-users with insights on factors that can guide their decision-making regarding optimizations that minimize ambient contamination, and metrics to assess data quality.
Keywords	Computational bioinformatics ; Transcriptomics ; Biology experimental methods ; Science ; Q
Subject code	310
Language	English
Publishing date	2023-07-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Comprehensive generation, visualization, and reporting of quality control metrics for single-cell RNA sequencing data

Rui Hong / Yusuke Koga / Shruthi Bandyadka / Anastasia Leshchyk / Yichen Wang / Vidya Akavoor / Xinyun Cao / Irzam Sarfraz / Zhe Wang / Salam Alabdullatif / Frederick Jansen / Masanao Yajima / W. Evan Johnson / Joshua D. Campbell

Nature Communications, Vol 13, Iss 1, Pp 1-

2022 Volume 9

Abstract: Quality control (QC) is a crucial step in single-cell RNA-seq data analysis. Here, the authors present the SCTK-QC pipeline which generates and visualizes a comprehensive set of QC metrics to streamline the process of detecting and removing poor quality ... ...

Abstract	Quality control (QC) is a crucial step in single-cell RNA-seq data analysis. Here, the authors present the SCTK-QC pipeline which generates and visualizes a comprehensive set of QC metrics to streamline the process of detecting and removing poor quality cells and other artifacts.
Keywords	Science ; Q
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Decontamination of ambient RNA in single-cell RNA-seq with DecontX

Shiyi Yang / Sean E. Corbett / Yusuke Koga / Zhe Wang / W Evan Johnson / Masanao Yajima / Joshua D. Campbell

Genome Biology, Vol 21, Iss 1, Pp 1-

2020 Volume 15

Abstract: Abstract Droplet-based microfluidic devices have become widely used to perform single-cell RNA sequencing (scRNA-seq). However, ambient RNA present in the cell suspension can be aberrantly counted along with a cell’s native mRNA and result in cross- ... ...

Abstract	Abstract Droplet-based microfluidic devices have become widely used to perform single-cell RNA sequencing (scRNA-seq). However, ambient RNA present in the cell suspension can be aberrantly counted along with a cell’s native mRNA and result in cross-contamination of transcripts between different cell populations. DecontX is a novel Bayesian method to estimate and remove contamination in individual cells. DecontX accurately predicts contamination levels in a mouse-human mixture dataset and removes aberrant expression of marker genes in PBMC datasets. We also compare the contamination levels between four different scRNA-seq protocols. Overall, DecontX can be incorporated into scRNA-seq workflows to improve downstream analyses.
Keywords	Bayesian mixture model ; Decontamination ; Single cell ; scRNA-seq ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
Language	English
Publishing date	2020-03-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Inactivation of the Hippo tumor suppressor pathway promotes melanoma

Marc A. Vittoria / Nathan Kingston / Kristyna Kotynkova / Eric Xia / Rui Hong / Lee Huang / Shayna McDonald / Andrew Tilston-Lunel / Revati Darp / Joshua D. Campbell / Deborah Lang / Xiaowei Xu / Craig J. Ceol / Xaralabos Varelas / Neil J. Ganem

Nature Communications, Vol 13, Iss 1, Pp 1-

2022 Volume 17

Abstract: Activating mutations of BRAF alone are inadequate to drive melanoma formation. Here the authors show that activation of Hippo signalling by oncogenic BRAF represents an additional safeguard to limit BRAF-dependent human melanocyte growth and melanoma ... ...

Abstract	Activating mutations of BRAF alone are inadequate to drive melanoma formation. Here the authors show that activation of Hippo signalling by oncogenic BRAF represents an additional safeguard to limit BRAF-dependent human melanocyte growth and melanoma formation.
Keywords	Science ; Q
Language	English
Publishing date	2022-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Smoking modulates different secretory subpopulations expressing SARS-CoV-2 entry genes in the nasal and bronchial airways

Scientific Reports, Vol 12, Iss 1, Pp 1-

2022 Volume 11

Abstract: Abstract SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in the airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial ... ...

Abstract	Abstract SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in the airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments have not been fully characterized using matched samples from large cohorts. Gene expression data from 793 nasal and 1673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking status (current versus former) was the only clinical factor significantly and reproducibly associated with VE gene expression. The expression of ACE2 and TMPRSS2 was higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the gene expression data confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose, these genes are not predominantly expressed in cell populations modulated by smoking. In individuals with elevated lung cancer risk, smoking-induced VE gene expression changes in the nose likely have minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2022-10-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Multi-modal profiling of human fetal liver hematopoietic stem cells reveals the molecular signature of engraftment

Nature Communications, Vol 13, Iss 1, Pp 1-

2022 Volume 13

Abstract: During human embryonic development, haematopoietic stem cells of the foetal liver undergo expansion, while retaining engraftment capacity. Here the authors apply CITE-seq to profile single cells from a human foetal liver, identifying a molecular ... ...

Abstract	During human embryonic development, haematopoietic stem cells of the foetal liver undergo expansion, while retaining engraftment capacity. Here the authors apply CITE-seq to profile single cells from a human foetal liver, identifying a molecular signature of engraftment potential
Keywords	Science ; Q
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The cellular and molecular determinants of emphysematous destruction in COPD

Masaru Suzuki / Marc A. Sze / Joshua D. Campbell / John F. Brothers / Marc E. Lenburg / John E. McDonough / W. Mark Elliott / Joel D. Cooper / Avrum Spira / James C. Hogg

Scientific Reports, Vol 7, Iss 1, Pp 1-

2017 Volume 9

Abstract: Abstract The introduction of microCT has made it possible to show that the terminal bronchioles are narrowed and destroyed before the onset of emphysematous destruction in COPD. This report extends those observations to the cellular and molecular level ... ...

Abstract	Abstract The introduction of microCT has made it possible to show that the terminal bronchioles are narrowed and destroyed before the onset of emphysematous destruction in COPD. This report extends those observations to the cellular and molecular level in the centrilobular phenotype of emphysematous destruction in lungs donated by persons with very severe COPD (n = 4) treated by lung transplantation with unused donor lungs (n = 4) serving as controls. These lung specimens provided companion samples to those previously examined by microCT (n = 61) that we examined using quantitative histology (n = 61) and gene expression profiling (n = 48). The histological analysis showed that remodeling and destruction of the bronchiolar and alveolar tissue is associated with macrophage, CD4, CD8, and B cell infiltration with increased formation of tertiary lymphoid organs. Moreover, gene set enrichment analysis showed that genes known to be expressed by natural killer (NK), lymphoid tissue inducer (LTi), and innate lymphoid cell 1 (ILC1) cells, but not ILC2 or ILC3 cells, were enriched in the expression profiles associated with CD4, CD8, and B cell infiltration. Based on these findings, we postulate that the centrilobular phenotype of emphysematous destruction COPD is driven by a Th1 response activated by infiltrating ILC1, NK, and LTi cells.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2017-08-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts.

Jennie Ong / Wim Timens / Vijay Rajendran / Arjan Algra / Avrum Spira / Marc E Lenburg / Joshua D Campbell / Maarten van den Berge / Dirkje S Postma / Anke van den Berg / Joost Kluiver / Corry-Anke Brandsma

PLoS ONE, Vol 12, Iss 9, p e

2017 Volume 0183815

Abstract: BACKGROUND:Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been ... ...

Abstract	BACKGROUND:Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function. MATERIALS AND METHODS:We investigated TGF-β1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays. TGF-β1-induced miRNA expression changes were validated and replicated in an independent set of lung fibroblasts composted of 10 controls and 15 COPD patients using qRT-PCR. Ago2-immunoprecipitation followed by mRNA expression profiling was used to identify the miRNA-targetomes of unstimulated and TGF-β1-stimulated primary lung fibroblasts (n = 2). The genes affected by TGF-β1-modulated miRNAs were identified by comparing the miRNA targetomes of unstimulated and TGF-β1-stimulated fibroblasts. RESULTS:Twenty-nine miRNAs were significantly differentially expressed after TGF-β1 stimulation (FDR<0.05). The TGF-β1-induced miR-455-3p and miR-21-3p expression changes were validated and replicated, with in addition, lower miR-455-3p levels in COPD (p<0.05). We identified 964 and 945 genes in the miRNA-targetomes of unstimulated and TGF-β1-stimulated lung fibroblasts, respectively. The TGF-β and Wnt pathways were significantly enriched among the Ago2-IP enriched and predicted targets of miR-455-3p and miR-21-3p. The miR-455-3p target genes HN1, NGF, STRADB, DLD and ANO3 and the miR-21-3p target genes HHEX, CHORDC1 and ZBTB49 were consistently more enriched after TGF-β1 stimulation. CONCLUSION:Two miRNAs, miR-455-3p and miR-21-3p, were induced by TGF-β1 in lung fibroblasts. The significant Ago2-IP enrichment of targets of these miRNAs related to the TGF-β and/or Wnt pathways (NGF, DLD, HHEX) in TGF-β1-stimulated fibroblasts suggest a ...
Keywords	Medicine ; R ; Science ; Q
Subject code	500
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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