*<ahref="https://orcid.org/0000-0001-7772-9578"target="orcid.widget"rel="noopener noreferrer"style="vertical-align:top;"><imgsrc="https://orcid.org/sites/default/files/images/orcid_16x16.png"style="width:1em;margin-right:.5em;"alt="ORCID iD icon">orcid.org/0000-0001-7772-9578</a>
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* PI Email: [douglas.strand@utsouthwestern.edu](mailto:douglas.strand@utsouthwestern.edu)
* PI Email: [douglas.strand@utsouthwestern.edu](mailto:douglas.strand@utsouthwestern.edu)
Data Analysis
Data Analysis
-------------
-------------
***Requirements:**
***Requirements:**
* 10x CellRanger analyzed data:
* 10x CellRanger analyzed data:
* "filtered\_gene\_bc\_matrice_mex" folder
* "filtered\_gene\_bc\_matrice_mex" folder
* csv file cellranger aggr used for aggregation
* csv file cellranger aggr used for aggregation
* demultiplex csv file to define subsets of samples
* demultiplex csv file to define subsets of samples
* R (v3.4.1) packages:
* R (v3.4.1) packages:
* methods (v3.4.1)
* methods (v3.4.1)
* optparse (v1.4.4)
* optparse (v1.4.4)
* Seurat (v2.3.1)
* Seurat (v2.3.1)
* readr (v1.1.1)
* readr (v1.1.1)
* fBasics (v3042.89)
* fBasics (v3042.89)
* pastecs (v1.3.21)
* pastecs (v1.3.21)
* qusage (v2.10.0)
* qusage (v2.10.0)
* RColorBrewer (v1.1-2)
* RColorBrewer (v1.1-2)
**and all dependencies*
**and all dependencies*
***Pipeline:**
***Pipeline:**
* Link cellranger count/aggr output to analysis
* Link cellranger count/aggr output to analysis
* Create demultiplex file to add custom sample groups
* Create demultiplex file to add custom sample groups
* Load R packages
* Load R packages
* Create analysis folders
* Create analysis folders
* Load analysis parameters (from default or overwrite from command line)
* Load analysis parameters (from default or overwrite from command line)
* Load cellranger data into R/Seurat
* Load cellranger data into R/Seurat
* Label cells based on their cell cycle stated using Seurat based method
* Label cells based on their cell cycle stated using Seurat based method
* Perform principle component analysis (PCA) using most highly variable genes (HVG) for downstream clustering etc
* Perform principle component analysis (PCA) using most highly variable genes (HVG) for downstream clustering etc
* Perform initial "over" clustering
* Perform initial "over" clustering
* Identify "highly stressed" cells using custom PCA based analysis, remove stressed clusters/cells, and re-cluster
* Identify "highly stressed" cells using custom PCA based analysis, remove stressed clusters/cells, and re-cluster
* Correlate cluster gene expression using Quantitative Set Analysis for Gene Expression (QuSAGE) on lineage genesets for identification (epithelia, and stroma)
* Correlate cluster gene expression using Quantitative Set Analysis for Gene Expression (QuSAGE) on lineage genesets for identification (epithelia, and stroma)
* Subset epithelia from stroma for additional analaysis
* Subset epithelia from stroma for additional analaysis
* Re-cluster cell types separately
* Re-cluster cell types separately
* Correlate cluster gene expression using QuSAGE on epithelial subtype genesets for identification (basal, luminal and "other")
* Correlate cluster gene expression using QuSAGE on epithelial subtype genesets for identification (basal, luminal and "other")
* Correlate cluster gene expression using QuSAGE on stromal subtype genesets for identification (fibroblasts, smooth muscle, endothelia and leukocyte)
* Correlate cluster gene expression using QuSAGE on stromal subtype genesets for identification (fibroblasts, smooth muscle, endothelia and leukocyte)
**Optional:* Correlate cluster gene expression using QuSAGE on additional genesets for analysis
**Optional:* Correlate cluster gene expression using QuSAGE on additional genesets for analysis
* Merge epithelial and stromal cells
* Merge epithelial and stromal cells
* Identify neuroendocrine cells from epithelial cells using custom PCA based analysis
* Identify neuroendocrine cells from epithelial cells using custom PCA based analysis
* Tabulate population cell numbers
* Tabulate population cell numbers
* Generate differentially expressed genelists (DEGs) of populations
* Generate differentially expressed genelists (DEGs) of populations
***Genesets:**
***Genesets:**
* Cell cycle:
* Cell cycle:
* "regev\_lab\_cell\_cycle\_genes.txt" G2M and S phase genes from [*Genome Res. 2015 Dec; 25(12): 1860–1872*](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665007/)
* "regev\_lab\_cell\_cycle\_genes.txt" G2M and S phase genes from [*Genome Res. 2015 Dec; 25(12): 1860–1872*](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665007/)
* Stress:
* Stress:
* "DEG\_C2.CGP.M10970.txt" MSigDB C2 Chemical and Genetic Perturbations M10970 [**CHUANG\_OXIDATIVE\_STRESS\_RESPONSE\_UP**](http://software.broadinstitute.org/gsea/msigdb/cards/CHUANG_OXIDATIVE_STRESS_RESPONSE_UP.html)
* "DEG\_C2.CGP.M10970.txt" MSigDB C2 Chemical and Genetic Perturbations M10970 [**CHUANG\_OXIDATIVE\_STRESS\_RESPONSE\_UP**](http://software.broadinstitute.org/gsea/msigdb/cards/CHUANG_OXIDATIVE_STRESS_RESPONSE_UP.html)
* "genes.deg.Stress.csv" DWS generated DEGs of stressed cells from scRNA-Seq of patient D17 only
* "genes.deg.Stress.csv" DWS generated DEGs of stressed cells from scRNA-Seq of patient D17 only
* "" DWS generated DEGs of stressed cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of stressed cells from scRNA-Seq of an aggregation of patient D17 and D27
* Lineage:
* Lineage:
* "DEG\_Epi_5FC.txt" DWS generated DEGs of epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_Epi_5FC.txt" DWS generated DEGs of epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_FMSt_5FC.txt" DWS generated DEGs of fibromuscular stroma from FACS population (bulk) RNA-sequencing
* "DEG\_FMSt_5FC.txt" DWS generated DEGs of fibromuscular stroma from FACS population (bulk) RNA-sequencing
* "genes.deg.Epi.csv" DWS generated DEGs of epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.Epi.csv" DWS generated DEGs of epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.St.csv" DWS generated DEGs of stromal cells from scRNA-Seq of patient D17 only
* "genes.deg.St.csv" DWS generated DEGs of stromal cells from scRNA-Seq of patient D17 only
* "" DWS generated DEGs of epithelial cells from from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of epithelial cells from from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of stromal cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of stromal cells from scRNA-Seq of an aggregation of patient D17 and D27
* Epithelia:
* Epithelia:
* "DEG\_BE_5FC.txt" DWS generated DEGs of basal epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_BE_5FC.txt" DWS generated DEGs of basal epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_LE_5FC.txt" DWS generated DEGs of luminal epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_LE_5FC.txt" DWS generated DEGs of luminal epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_OE_5FC.txt" DWS generated DEGs of "other" epithelia from FACS population (bulk) RNA-sequencing
* "DEG\_OE_5FC.txt" DWS generated DEGs of "other" epithelia from FACS population (bulk) RNA-sequencing
* "genes.deg.BE.csv" DWS generated DEGs of basal epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.BE.csv" DWS generated DEGs of basal epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.LE.csv" DWS generated DEGs of luminal epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.LE.csv" DWS generated DEGs of luminal epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.OE1.csv" DWS generated DEGs of "other" epithelia cluster 1 cells from scRNA-Seq of patient D17 only
* "genes.deg.OE1.csv" DWS generated DEGs of "other" epithelia cluster 1 cells from scRNA-Seq of patient D17 only
* "genes.deg.OE2.csv" DWS generated DEGs of "other" epithelia cluster 2 cells from scRNA-Seq of patient D17 only
* "genes.deg.OE2.csv" DWS generated DEGs of "other" epithelia cluster 2 cells from scRNA-Seq of patient D17 only
* "" DWS generated DEGs of basal epithelial cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of basal epithelial cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of luminal epithelial cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of luminal epithelial cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of "other" epithelia cluster 1 cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of "other" epithelia cluster 1 cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of "other" epithelia cluster 2 cells from scRNA-Seq of an aggregation of patient D17 and D27
* "" DWS generated DEGs of "other" epithelia cluster 2 cells from scRNA-Seq of an aggregation of patient D17 and D27
* Stroma:
* Stroma:
* "DEG\_C5.BP.M11704.txt" MSigDB C5 GO Biological Processes M11704 [**GO\_ENDOTHELIAL\_CELL\_DIFFERENTIATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_ENDOTHELIAL_CELL_DIFFERENTIATION.html)
* "DEG\_C5.BP.M11704.txt" MSigDB C5 GO Biological Processes M11704 [**GO\_ENDOTHELIAL\_CELL\_DIFFERENTIATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_ENDOTHELIAL_CELL_DIFFERENTIATION.html)
* "DEG\_C5.BP.M10794.txt" MSigDB C5 GO Biological Processes M10794 [**GO\_SMOOTH\_MUSCLE\_CELL\_DIFFERENTIATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_SMOOTH_MUSCLE_CELL_DIFFERENTIATION.html)
* "DEG\_C5.BP.M10794.txt" MSigDB C5 GO Biological Processes M10794 [**GO\_SMOOTH\_MUSCLE\_CELL\_DIFFERENTIATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_SMOOTH_MUSCLE_CELL_DIFFERENTIATION.html)
* "DEG\_C5.BP.M13024.txt" MSigDB C5 GO Biological Processes M13024 [**GO\_REGULATION\_OF\_FIBROBLAST\_PROLIFERATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_REGULATION_OF_FIBROBLAST_PROLIFERATION.html)
* "DEG\_C5.BP.M13024.txt" MSigDB C5 GO Biological Processes M13024 [**GO\_REGULATION\_OF\_FIBROBLAST\_PROLIFERATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_REGULATION_OF_FIBROBLAST_PROLIFERATION.html)
* "DEG\_C5.BP.M10124.txt" MSigDB C5 GO Biological Processes M10124 [**GO\_LEUKOCYTE\_ACTIVATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_LEUKOCYTE_ACTIVATION.html)
* "DEG\_C5.BP.M10124.txt" MSigDB C5 GO Biological Processes M10124 [**GO\_LEUKOCYTE\_ACTIVATION**](http://software.broadinstitute.org/gsea/msigdb/cards/GO_LEUKOCYTE_ACTIVATION.html)
* Neuroendocrine:
* Neuroendocrine:
* "EurUrol.2005.NE.txt" Neuroendocrine markers from Table 1 of [*Eur Urol. 2005 Feb;47(2):147-55*](https://www.ncbi.nlm.nih.gov/pubmed/15661408)
* "EurUrol.2005.NE.txt" Neuroendocrine markers from Table 1 of [*Eur Urol. 2005 Feb;47(2):147-55*](https://www.ncbi.nlm.nih.gov/pubmed/15661408)
* "genes.deg.NE.csv" DWS generated DEGs of neuroendocrine epithelial cells from scRNA-Seq of patient D17 only
* "genes.deg.NE.csv" DWS generated DEGs of neuroendocrine epithelial cells from scRNA-Seq of patient D17 only
* "" DWS generated DEGs of neuroendocrine epithelial cells from scRNA-Seq of patient D17 only
* "" DWS generated DEGs of neuroendocrine epithelial cells from scRNA-Seq of patient D17 only
* Lung epithelia from [Lung Gene Expression Analysis (LGEA) Web Portal](https://research.cchmc.org/pbge/lunggens/mainportal.html):
* Lung epithelia from [Lung Gene Expression Analysis (LGEA) Web Portal](https://research.cchmc.org/pbge/lunggens/mainportal.html):
* "Basal cells-signature-genes.csv" scRNA-Seq LGEA generated top 20 DEGs for [human lung Basal Cells] (https://research.cchmc.org/pbge/lunggens/lungDisease/celltype_IPF.html?cid=3)
* "Basal cells-signature-genes.csv" scRNA-Seq LGEA generated top 20 DEGs for [human lung Basal Cells] (https://research.cchmc.org/pbge/lunggens/lungDisease/celltype_IPF.html?cid=3)
* "Normal AT2 cells-signature-genes.csv" scRNA-Sequecing LGEA generated top 20 DEGs for [human lung Alveolar Type 2 Cells](https://research.cchmc.org/pbge/lunggens/lungDisease/celltype_IPF.html?cid=1)
* "Normal AT2 cells-signature-genes.csv" scRNA-Sequecing LGEA generated top 20 DEGs for [human lung Alveolar Type 2 Cells](https://research.cchmc.org/pbge/lunggens/lungDisease/celltype_IPF.html?cid=1)
* "Club\_Goblet cells-signature-genes.csv" scRNA-Sequencing LGEA generated top 20 DEGs for [human lung Club/Goblet Cells](https://research.cchmc.org/pbge/lunggens/lungDisease/celltype_IPF.html?cid=4)
* "Club\_Goblet cells-signature-genes.csv" scRNA-Sequencing LGEA generated top 20 DEGs for [human lung Club/Goblet Cells](https://research.cchmc.org/pbge/lunggens/lungDisease/celltype_IPF.html?cid=4)