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annotate Seurat.R @ 2:321bdd834266 draft

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"planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
author iuc
date Thu, 19 Dec 2019 02:42:56 -0500
parents 7319f83ae734
children 764f076e9d52
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7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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1 #' ---
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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2 #' title: "Seurat Analysis"
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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3 #' author: "Performed using Galaxy"
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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4 #' params:
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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5 #' counts: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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6 #' min_cells: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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7 #' min_genes: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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8 #' low_thresholds: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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9 #' high_thresholds: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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10 #' numPCs: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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11 #' cells_use: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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12 #' resolution: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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13 #' min_pct: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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14 #' logfc_threshold: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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15 #' showcode: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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16 #' warn: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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17 #' varstate: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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18 #' vlnfeat: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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19 #' featplot: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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20 #' PCplots: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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21 #' tsne: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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22 #' heatmaps: ""
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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23 #' ---
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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25 #+ echo=F, warning = F, message=F
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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26 options(show.error.messages = F, error = function(){cat(geterrmessage(), file = stderr()); q("no", 1, F)})
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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27 showcode <- as.logical(params$showcode)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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28 warn <- as.logical(params$warn)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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29 varstate <- as.logical(params$varstate)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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30 vlnfeat <- as.logical(params$vlnfeat)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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31 featplot <- as.logical(params$featplot)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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32 PCplots <- as.logical(params$PCplots)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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33 tsne <- as.logical(params$tsne)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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34 heatmaps <- as.logical(params$heatmaps)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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35
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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36 # we need that to not crash Galaxy with an UTF-8 error on German LC settings.
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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37 loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8")
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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38
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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39
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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40 #+ echo = F, warning = `warn`, include =`varstate`
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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41 min_cells <- as.integer(params$min_cells)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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42 min_genes <- as.integer(params$min_genes)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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43 low_thresholds <- as.integer(params$low_thresholds)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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44 high_thresholds <- as.integer(params$high_thresholds)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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45 numPCs <- as.integer(params$numPCs)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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46 cells_use <- as.integer(params$cells_use)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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47 resolution <- as.double(params$resolution)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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48 min_pct <- as.double(params$min_pct)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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49 logfc_threshold <- as.double(params$logfc_thresh)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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50 print(paste0("Minimum cells: ", min_cells))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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51 print(paste0("Minimum features: ", min_genes))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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52 print(paste0("Umi low threshold: ", low_thresholds))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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53 print(paste0("Umi high threshold: ", high_thresholds))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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54 print(paste0("Number of principal components: ", numPCs))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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55 print(paste0("Resolution: ", resolution))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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56 print(paste0("Minimum percent of cells", min_pct))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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57 print(paste0("Logfold change threshold", logfc_threshold))
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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58
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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59 #+ echo = FALSE
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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60 if(showcode == TRUE) print("Read in data, generate inital Seurat object")
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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61 #+ echo = `showcode`, warning = `warn`, message = F
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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62 counts <- read.delim(params$counts, row.names = 1)
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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63 seuset <- Seurat::CreateSeuratObject(counts = counts, min.cells = min_cells, min.features = min_genes)
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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64
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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65 #+ echo = FALSE
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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66 if(showcode == TRUE && vlnfeat == TRUE) print("Raw data vizualization")
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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67 #+ echo = `showcode`, warning = `warn`, include=`vlnfeat`
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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68 Seurat::VlnPlot(object = seuset, features = c("nFeature_RNA", "nCount_RNA"))
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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69 Seurat::FeatureScatter(object = seuset, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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70
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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71 #+ echo = FALSE
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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72 if(showcode == TRUE) print("Filter and normalize for UMI counts")
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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73 #+ echo = `showcode`, warning = `warn`
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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74 seuset <- subset(seuset, subset = `nCount_RNA` > low_thresholds & `nCount_RNA` < high_thresholds)
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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75 seuset <- Seurat::NormalizeData(seuset, normalization.method = "LogNormalize", scale.factor = 10000)
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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76
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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77 #+ echo = FALSE
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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78 if(showcode == TRUE && featplot == TRUE) print("Variable Genes")
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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79 #+ echo = `showcode`, warning = `warn`, include = `featplot`
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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80 seuset <- Seurat::FindVariableFeatures(object = seuset, selection.method = "mvp")
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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81 Seurat::VariableFeaturePlot(seuset, cols = c("black", "red"), selection.method = "disp")
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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82 seuset <- Seurat::ScaleData(object = seuset, vars.to.regress = "nCount_RNA")
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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83
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
iuc
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84 #+ echo = FALSE
2
321bdd834266 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 3cf715ec11e2c9944f46572e324e5b2db5aa151f"
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85 if(showcode == TRUE && PCplots == TRUE) print("PCA Visualization")
1
7319f83ae734 "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/seurat commit 88cf23c767023f71b4ea1e72aac568cc694cc34a"
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86 #+ echo = `showcode`, warning = `warn`, include = `PCplots`
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87 seuset <- Seurat::RunPCA(seuset, npcs = numPCs)
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88 Seurat::VizDimLoadings(seuset, dims = 1:2)
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89 Seurat::DimPlot(seuset, dims = c(1,2), reduction = "pca")
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90 Seurat::DimHeatmap(seuset, dims = 1:numPCs, nfeatures = 30, reduction = "pca")
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91 seuset <- Seurat::JackStraw(seuset, dims=numPCs, reduction = "pca", num.replicate = 100)
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92 seuset <- Seurat::ScoreJackStraw(seuset, dims = 1:numPCs)
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93 Seurat::JackStrawPlot(seuset, dims = 1:numPCs)
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94 Seurat::ElbowPlot(seuset, ndims = numPCs, reduction = "pca")
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95
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96 #+ echo = FALSE
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97 if(showcode == TRUE && tsne == TRUE) print("tSNE")
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98 #+ echo = `showcode`, warning = `warn`, include = `tsne`
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99 seuset <- Seurat::FindNeighbors(object = seuset)
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100 seuset <- Seurat::FindClusters(object = seuset)
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101 seuset <- Seurat::RunTSNE(seuset, dims = 1:numPCs, resolution = resolution)
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102 Seurat::DimPlot(seuset, reduction = "tsne")
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103
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104 #+ echo = FALSE
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105 if(showcode == TRUE && heatmaps == TRUE) print("Marker Genes")
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106 #+ echo = `showcode`, warning = `warn`, include = `heatmaps`
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107 markers <- Seurat::FindAllMarkers(seuset, only.pos = TRUE, min.pct = min_pct, logfc.threshold = logfc_threshold)
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108 top10 <- dplyr::group_by(markers, cluster)
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109 top10 <- dplyr::top_n(top10, 10, avg_logFC)
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110 Seurat::DoHeatmap(seuset, features = top10$gene)