Last updated: 2021-07-04

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Knit directory: Embryoid_Body_Pilot_Workflowr/analysis/

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/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/output/mergedObjects/Harmony.Batchindividual.rds ../output/mergedObjects/Harmony.Batchindividual.rds
/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/data/dge/hESC1.rawdge.txt.gz ../data/dge/hESC1.rawdge.txt.gz
/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/data/dge/iPS-to-EB_20Day_dge.txt.gz ../data/dge/iPS-to-EB_20Day_dge.txt.gz
/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/output/CaoEtAl.Obj.CellsOfAllClusters.ProteinCodingGenes.rds ../output/CaoEtAl.Obj.CellsOfAllClusters.ProteinCodingGenes.rds
/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/output/TranferredAnnotations_ReferenceInt_JustMeso.csv ../output/TranferredAnnotations_ReferenceInt_JustMeso.csv

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library(Seurat)
library(edgeR)
Loading required package: limma
library(dplyr)

Attaching package: 'dplyr'
The following objects are masked from 'package:stats':

    filter, lag
The following objects are masked from 'package:base':

    intersect, setdiff, setequal, union
library(tidyr)
library(tibble)
library(purrr)
library(harmony)
Loading required package: Rcpp
library(ggplot2)

loading data

first, my data

merged<- readRDS("/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/output/mergedObjects/Harmony.Batchindividual.rds")

subset to only mesoderm cells (clusters 2 and 6, res 0.1)

merged<- subset(merged, idents = c(2,6))
DimPlot(merged)

loading in hESC and iPS-to-EB raw dges from scHCL reference set

hESC<- read.table("/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/data/dge/hESC1.rawdge.txt.gz", header=T, row.names = 1)

iPStoEBday20<- read.table("/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/data/dge/iPS-to-EB_20Day_dge.txt.gz", header=T, row.names = 1)

Note: there is no available metadata for these iPS to EB differentiations (no cell annotations online)

make a seurat objects with all of the data

hESC.obj<- CreateSeuratObject(hESC)
Warning: Feature names cannot have underscores ('_'), replacing with dashes
('-')
EB20.obj<- CreateSeuratObject(iPStoEBday20)
Warning: Feature names cannot have underscores ('_'), replacing with dashes
('-')
#normalizing each dataset
hESC.obj<- suppressWarnings(SCTransform(hESC.obj, variable.features.n=5000,verbose=F))

EB20.obj<-suppressWarnings(SCTransform(EB20.obj, variable.features.n=5000,verbose=F))
Cao.obj<-readRDS("/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/output/CaoEtAl.Obj.CellsOfAllClusters.ProteinCodingGenes.rds")
Cao.obj<- RunPCA(Cao.obj, npcs= 100, verbose = F)

Cao.obj<- FindNeighbors(Cao.obj, dims = 1:30, verbose = F)
Cao.obj<- RunUMAP(Cao.obj, dims=1:30, verbose = F)
Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
This message will be shown once per session
#rename Cao metadata so none match with EB (just need to replace Batch)
colnames(Cao.obj@meta.data)
 [1] "orig.ident"        "nCount_RNA"        "nFeature_RNA"     
 [4] "Assay"             "Batch"             "Experiment_batch" 
 [7] "Main_cluster_name" "Fetus_id"          "Sex"              
[10] "nCount_SCT"        "nFeature_SCT"     
colnames(Cao.obj@meta.data)[5]<-"Batch_week"
#rename orig.idents
hESC.obj$orig.ident<- "scHCL.hESC"

EB20.obj$orig.ident<- "scHCL.EB20"
merged$orig.ident<- "EB.Pilot"
Cao.obj$orig.ident<- "Cao.EtAl"
#merge objects
obj.list<- list(Cao.obj, merged, hESC.obj, EB20.obj)
merge.all<- merge(x=obj.list[[1]], y=c(obj.list[[2]], obj.list[[3]], obj.list[[4]]), merge.data=T)
FeatureScatter(merge.all, feature1 = "nCount_SCT", feature2 = "nFeature_SCT", group.by = "orig.ident")

FeatureScatter(merge.all, feature1 = "nCount_RNA", feature2 = "nFeature_RNA", group.by = "orig.ident")

merge.all<- SCTransform(merge.all, variable.features.n = 5000, vars.to.regress = c("orig.ident"), assay= "SCT")

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  |=========                                                             |  12%
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all.genes= rownames(merge.all)
merge.all<-FindVariableFeatures(merge.all,selection.method="vst", nfeatures = 5000)
merge.all<- ScaleData(merge.all, features = all.genes, assay = "SCT")
Centering and scaling data matrix
merge.all<-RunPCA(merge.all, npcs = 100, verbose=F, Assay="SCT")
DimPlot(merge.all, reduction = "pca", group.by = "orig.ident")

merge.all<- RunHarmony(merge.all, c("orig.ident", "individual", "Batch"), theta = c(3,1,1), plot_convergence = T, assay.use = "SCT")
Harmony 1/10
Harmony 2/10
Harmony 3/10
Harmony 4/10
Harmony 5/10
Harmony converged after 5 iterations
Warning: Invalid name supplied, making object name syntactically valid. New
object name is Seurat..ProjectDim.SCT.harmony; see ?make.names for more details
on syntax validity

DimPlot(merge.all, group.by= "orig.ident", reduction= "harmony")

merge.all<- RunUMAP(merge.all,dims=1:100, reduction="harmony")
16:21:11 UMAP embedding parameters a = 0.9922 b = 1.112
16:21:11 Read 49629 rows and found 100 numeric columns
16:21:11 Using Annoy for neighbor search, n_neighbors = 30
16:21:11 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
16:21:30 Writing NN index file to temp file /tmp/jobs/12028895/Rtmp2UCfIN/file3b20e26095e4a
16:21:30 Searching Annoy index using 1 thread, search_k = 3000
16:22:00 Annoy recall = 96.24%
16:22:01 Commencing smooth kNN distance calibration using 1 thread
16:22:04 Initializing from normalized Laplacian + noise
16:22:11 Commencing optimization for 200 epochs, with 2302498 positive edges
16:23:17 Optimization finished
V<- DimPlot(merge.all, group.by = "Main_cluster_name", label = T, label.size = 2.5, repel = T)+NoLegend()
Warning: Using `as.character()` on a quosure is deprecated as of rlang 0.3.0.
Please use `as_label()` or `as_name()` instead.
This warning is displayed once per session.
V
Warning: ggrepel: 9 unlabeled data points (too many overlaps). Consider
increasing max.overlaps

Add new metadata to include Main_cluster_name as well as cluster labels from EB Pilot DE results, and orig ident of the HCL data

merge.all<- AddMetaData(merge.all, col.name = "all.labels", metadata = merge.all@meta.data$Main_cluster_name)

#for cells with NA for main_cluster_name, replace label from SCT_snn_res 0.1
for (i in (1:nrow(merge.all@meta.data))){
if (is.na(merge.all@meta.data$all.labels[i]) == T){
  merge.all@meta.data$all.labels[i]<- merge.all@meta.data$SCT_snn_res.0.1[i]
}
}

#and now replace remaining NAs with orig.ident
for (i in (1:nrow(merge.all@meta.data))){
if (is.na(merge.all@meta.data$all.labels[i]) == T){
  merge.all@meta.data$all.labels[i]<- merge.all@meta.data$orig.ident[i]
}
}
V<- DimPlot(merge.all, group.by = "all.labels", label = T, label.size = 2.5, repel = T)+NoLegend()

V
Warning: ggrepel: 9 unlabeled data points (too many overlaps). Consider
increasing max.overlaps

V<- DimPlot(merge.all, group.by = "orig.ident", label = F, order= c("scHCL.hESC","scHCL.EB20", "EB.Pilot","Cao.EtAl"))

V

DimPlot(merge.all, split.by = "orig.ident", label = F, order= c("scHCL.hESC","scHCL.EB20", "EB.Pilot","Cao.EtAl"))

options(ggrepel.max.overlaps = Inf)
#subset object to just my data and Cao reference, plot UMAP
Idents(merge.all)<- "orig.ident"
sub<- subset(merge.all, idents= c("Cao.EtAl", "EB.Pilot"))
V<- DimPlot(sub, group.by = "all.labels", label = T, repel = T, label.size = 2.5)+NoLegend()
V

V<- DimPlot(sub, group.by = "Main_cluster_name", label = T, repel = T, label.size = 2.5)+NoLegend()
V

V<- DimPlot(sub, group.by = "SCT_snn_res.0.1", label = T, repel = T, label.size = 2.5)+NoLegend()
V

DimPlot(sub, group.by = "orig.ident")

DimPlot(sub, group.by = "orig.ident", order="Cao.EtAl")

#Subset object to just my data and HCL references, plot UMAP
sub<- subset(merge.all, idents= c("scHCL.EB20", "EB.Pilot"))
V<- DimPlot(sub, group.by = "orig.ident", label = F)
V

sub<- subset(merge.all, idents= c("scHCL.EB20", "EB.Pilot", "Cao.EtAl"))
DimPlot(sub, group.by="orig.ident", pt.size = 0.2, label=F) 

DimPlot(merge.all, split.by="orig.ident",group.by = "all.labels", pt.size = 0.2, label=F) +NoLegend()

Now, will transfer labels for Cao Et Al and hESC onto my data.

#subset to remove scHCL.EB20
sub<- subset(merge.all, idents= c("Cao.EtAl", "EB.Pilot", "scHCL.hESC"))
#compute distance matrix based on harmony embeddings, dims 1:100
har_embeds<- sub@reductions$harmony@cell.embeddings

har_distmat<- as.matrix(dist(har_embeds, method="euclidean", upper=TRUE))
#vectors with cell ids from Cao.EtAl, EB.pilot, and scHCL.hESC
EB.pilot.ids<-rownames(merge.all@meta.data[merge.all@meta.data$orig.ident == "EB.Pilot",])

#subset rows to only cells in EB.pilot
sub_har_distmat<- har_distmat[rownames(har_distmat) %in% EB.pilot.ids,]

#subset cols to only cells not in EB.pilot
'%notin%'<- Negate('%in%')
sub_har_distmat<- sub_har_distmat[,colnames(sub_har_distmat) %notin% EB.pilot.ids]
nearest.ref.cell.id<- NULL
nearest.ref.cell.dist<- NULL
#for loop, loop through each row
for (i in 1:nrow(sub_har_distmat)){
  nearest.ref.cell.dist[i]<- min(sub_har_distmat[i,])
  nearest.ref.cell.id[i]<- names(which.min(sub_har_distmat[i,]))
}

nearest.ref.table<- cbind(rownames(sub_har_distmat), nearest.ref.cell.id,nearest.ref.cell.dist)

colnames(nearest.ref.table)<- c("EB.cell.id", "nearest.ref.cell.id", "harmony.dist.to.nearest.ref.cell")
#add annotation
ann<- as.data.frame(merge.all@meta.data$all.labels)
ann<- cbind(rownames(merge.all@meta.data), ann)
colnames(ann)<- c("nearest.ref.cell.id", "annotation")

nearest.ref.table<- as.data.frame(nearest.ref.table)
nearest.ann<- left_join(nearest.ref.table, ann, by=c("nearest.ref.cell.id"))
a<- as.data.frame(table(nearest.ann$annotation))
a<- a[a$Var1 != "scHCL.EB20",]
a<- a[a$Var1 != "0",]
a<- a[a$Var1 != "1",]
a<- a[a$Var1 != "2",]
a<- a[a$Var1 != "3",]
a<- a[a$Var1 != "4",]
a<- a[a$Var1 != "5",]
a<- a[a$Var1 != "6",]
a<- a[a$Var1 != "7",]
colnames(a)<- c("reference.cell.type", "Frequency")

a
                                 reference.cell.type Frequency
3                             AFP_ALB positive cells         0
4                                       Acinar cells       396
5                               Adrenocortical cells         2
6                                     Amacrine cells         0
7                           Antigen presenting cells         4
8                                         Astrocytes         0
9                                      Bipolar cells         0
10         Bronchiolar and alveolar epithelial cells         0
11                        CCL19_CCL21 positive cells         0
12                          CLC_IL5RA positive cells        29
13                          CSH1_CSH2 positive cells         1
14                                    Cardiomyocytes       235
15                                  Chromaffin cells         0
16                         Ciliated epithelial cells         9
17         Corneal and conjunctival epithelial cells         0
18                                      Ductal cells         0
19                         ELF3_AGBL2 positive cells        37
20                                          ENS glia         3
21                                       ENS neurons         0
22                                 Endocardial cells        43
23                              Epicardial fat cells         0
24                                     Erythroblasts       250
25                                Excitatory neurons         0
26                         Extravillous trophoblasts         1
27                                    Ganglion cells         0
28                                      Goblet cells         0
29                                   Granule neurons         0
30                          Hematopoietic stem cells        38
31                                      Hepatoblasts        27
32                                  Horizontal cells         0
33                        IGFBP1_DKK1 positive cells       268
34                           Inhibitory interneurons         0
35                                Inhibitory neurons         0
36                       Intestinal epithelial cells        20
37                             Islet endocrine cells         0
38                                  Lens fibre cells         0
39                             Limbic system neurons         0
40                       Lymphatic endothelial cells        30
41                                    Lymphoid cells        12
42                        MUC13_DMBT1 positive cells        17
43                                    Megakaryocytes        37
44                                   Mesangial cells        48
45                                 Mesothelial cells         8
46                                 Metanephric cells       119
47                                         Microglia        16
48                                     Myeloid cells         4
49                              Neuroendocrine cells         4
50                                  Oligodendrocytes         2
51                         PAEP_MECOM positive cells         0
52                     PDE11A_FAM19A2 positive cells         0
53                        PDE1C_ACSM3 positive cells         0
54                          Parietal and chief cells        22
55                               Photoreceptor cells         0
56                                  Purkinje neurons         0
57                             Retinal pigment cells         1
58               Retinal progenitors and Muller glia         3
59                        SATB2_LRRC7 positive cells         3
60                        SKOR2_NPSR1 positive cells         0
61                      SLC24A4_PEX5L positive cells         1
62                       SLC26A4_PAEP positive cells         0
63                          STC2_TLX1 positive cells         1
64                                   Satellite cells        48
65                                     Schwann cells         7
66                             Skeletal muscle cells        43
67                               Smooth muscle cells        25
68                         Squamous epithelial cells        11
69                                    Stellate cells       110
70                                     Stromal cells         5
71                                    Sympathoblasts         0
72 Syncytiotrophoblasts and villous cytotrophoblasts         0
73                           Thymic epithelial cells         0
74                                        Thymocytes         0
75                           Trophoblast giant cells        21
76                              Unipolar brush cells         0
77                                Ureteric bud cells         0
78                        Vascular endothelial cells       105
79                                  Visceral neurons         0
81                                        scHCL.hESC      1315
sub<- subset(merge.all, idents= c("EB.Pilot"))
EB.cell.id<- rownames(sub@meta.data)
sub@meta.data<- cbind(sub@meta.data, EB.cell.id)
sub@meta.data<- full_join(sub@meta.data, nearest.ann, by= c("EB.cell.id"))
rownames(sub@meta.data)<- EB.cell.id

V<- DimPlot(sub, group.by="annotation", pt.size = 0.2, label.size = 2.5,label=T, repel=T) +NoLegend()

V

mostcommon.ann<- NULL
maxann.FIVEnearest<- NULL

#for loop, loop through each row
for (i in 1:nrow(sub_har_distmat)){
  cell<- sub_har_distmat[i,]
  cell<- cell[order(cell)]
  topfive<- names(cell[1:5])
  
  #get the annotations of the nearest 5 reference cells
  topfiveann<- merge.all@meta.data$all.labels[rownames(merge.all@meta.data) %in% topfive]
  
  #if/else at least 3/5 match annotations
  maxann<- max(table(topfiveann))
  finalann<- names(which.max(table(topfiveann)))
  
  maxann.FIVEnearest[i]<- maxann
  
  if(maxann >= 3){
    mostcommon.ann[i]<- finalann
  } else {
    mostcommon.ann[i]<- "uncertain"
  }
  
}

CommonAnnDF<- as.data.frame(cbind(rownames(sub_har_distmat), mostcommon.ann, maxann.FIVEnearest))
colnames(CommonAnnDF)<- c("EB.cell.id", "Annotation", "NoutofFIVErefneighborsWithSameAnnotation")
write.csv(CommonAnnDF,"/project2/gilad/katie/Pilot_HumanEBs/Embryoid_Body_Pilot_Workflowr/output/TranferredAnnotations_ReferenceInt_JustMeso.csv")
b<- table(CommonAnnDF$Annotation)

b

               Acinar cells    Antigen presenting cells 
                        390                           1 
   CLC_IL5RA positive cells              Cardiomyocytes 
                          4                         241 
  Ciliated epithelial cells   ELF3_AGBL2 positive cells 
                         10                           4 
                   ENS glia           Endocardial cells 
                          1                          40 
              Erythroblasts    Hematopoietic stem cells 
                        254                          45 
               Hepatoblasts  IGFBP1_DKK1 positive cells 
                         22                         269 
Intestinal epithelial cells Lymphatic endothelial cells 
                          7                          24 
             Lymphoid cells  MUC13_DMBT1 positive cells 
                          2                          23 
             Megakaryocytes             Mesangial cells 
                         31                          23 
          Metanephric cells                   Microglia 
                         84                          10 
              Myeloid cells    Parietal and chief cells 
                          1                           5 
              Schwann cells       Skeletal muscle cells 
                          1                           7 
        Smooth muscle cells   Squamous epithelial cells 
                         17                           9 
             Stellate cells  Vascular endothelial cells 
                        115                          98 
                 scHCL.hESC                   uncertain 
                       1289                         354 
#print fetal celltypes not present in EB data
unique(merge.all@meta.data$Main_cluster_name)[unique(merge.all@meta.data$Main_cluster_name) %notin% names(b)] 
 [1] "Retinal progenitors and Muller glia"              
 [2] "Ganglion cells"                                   
 [3] "Retinal pigment cells"                            
 [4] "Horizontal cells"                                 
 [5] "Photoreceptor cells"                              
 [6] "Lens fibre cells"                                 
 [7] "Amacrine cells"                                   
 [8] "Corneal and conjunctival epithelial cells"        
 [9] "PDE11A_FAM19A2 positive cells"                    
[10] "Bipolar cells"                                    
[11] "Stromal cells"                                    
[12] "Adrenocortical cells"                             
[13] "Chromaffin cells"                                 
[14] "Sympathoblasts"                                   
[15] "SLC26A4_PAEP positive cells"                      
[16] "CSH1_CSH2 positive cells"                         
[17] "Inhibitory interneurons"                          
[18] "SLC24A4_PEX5L positive cells"                     
[19] "Purkinje neurons"                                 
[20] "Astrocytes"                                       
[21] "Granule neurons"                                  
[22] "Oligodendrocytes"                                 
[23] "Unipolar brush cells"                             
[24] "SKOR2_NPSR1 positive cells"                       
[25] "Excitatory neurons"                               
[26] "Inhibitory neurons"                               
[27] "Limbic system neurons"                            
[28] "SATB2_LRRC7 positive cells"                       
[29] "Epicardial fat cells"                             
[30] "Visceral neurons"                                 
[31] "Mesothelial cells"                                
[32] "ENS neurons"                                      
[33] "Ureteric bud cells"                               
[34] "Neuroendocrine cells"                             
[35] "Bronchiolar and alveolar epithelial cells"        
[36] "Satellite cells"                                  
[37] "CCL19_CCL21 positive cells"                       
[38] "Ductal cells"                                     
[39] "Islet endocrine cells"                            
[40] "AFP_ALB positive cells"                           
[41] "Trophoblast giant cells"                          
[42] "Extravillous trophoblasts"                        
[43] "PAEP_MECOM positive cells"                        
[44] "Syncytiotrophoblasts and villous cytotrophoblasts"
[45] "STC2_TLX1 positive cells"                         
[46] "PDE1C_ACSM3 positive cells"                       
[47] "Goblet cells"                                     
[48] "Thymic epithelial cells"                          
[49] "Thymocytes"                                       
[50] NA                                                 
sub<- subset(merge.all, idents= c("EB.Pilot"))
EB.cell.id<- rownames(sub@meta.data)
sub@meta.data<- cbind(sub@meta.data, EB.cell.id)
sub@meta.data<- full_join(sub@meta.data, CommonAnnDF, by= c("EB.cell.id"))
rownames(sub@meta.data)<- EB.cell.id

V<- DimPlot(sub, group.by="Annotation", pt.size = 0.2, label.size = 2.5,label=T, repel=T) +NoLegend()

V

sessionInfo()
R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
[1] C

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] ggplot2_3.3.3   harmony_1.0     Rcpp_1.0.6      purrr_0.3.4    
 [5] tibble_3.0.4    tidyr_1.1.0     dplyr_1.0.2     edgeR_3.28.1   
 [9] limma_3.42.2    Seurat_3.2.0    workflowr_1.6.2

loaded via a namespace (and not attached):
  [1] Rtsne_0.15            colorspace_2.0-0      deldir_0.1-28        
  [4] ellipsis_0.3.1        ggridges_0.5.2        rprojroot_2.0.2      
  [7] fs_1.4.2              spatstat.data_1.4-3   farver_2.0.3         
 [10] leiden_0.3.3          listenv_0.8.0         npsurv_0.4-0         
 [13] ggrepel_0.9.0         RSpectra_0.16-0       codetools_0.2-16     
 [16] splines_3.6.1         lsei_1.2-0            knitr_1.29           
 [19] polyclip_1.10-0       jsonlite_1.7.2        ica_1.0-2            
 [22] cluster_2.1.0         png_0.1-7             uwot_0.1.10          
 [25] shiny_1.5.0           sctransform_0.2.1     compiler_3.6.1       
 [28] httr_1.4.2            Matrix_1.2-18         fastmap_1.0.1        
 [31] lazyeval_0.2.2        later_1.1.0.1         htmltools_0.5.0      
 [34] tools_3.6.1           rsvd_1.0.3            igraph_1.2.6         
 [37] gtable_0.3.0          glue_1.4.2            RANN_2.6.1           
 [40] reshape2_1.4.4        rappdirs_0.3.3        spatstat_1.64-1      
 [43] vctrs_0.3.6           gdata_2.18.0          ape_5.4-1            
 [46] nlme_3.1-140          lmtest_0.9-37         xfun_0.16            
 [49] stringr_1.4.0         globals_0.12.5        mime_0.9             
 [52] miniUI_0.1.1.1        lifecycle_0.2.0       irlba_2.3.3          
 [55] gtools_3.8.2          goftest_1.2-2         future_1.18.0        
 [58] MASS_7.3-51.4         zoo_1.8-8             scales_1.1.1         
 [61] promises_1.1.1        spatstat.utils_1.17-0 parallel_3.6.1       
 [64] RColorBrewer_1.1-2    yaml_2.2.1            reticulate_1.20      
 [67] pbapply_1.4-2         gridExtra_2.3         rpart_4.1-15         
 [70] stringi_1.5.3         highr_0.8             caTools_1.18.0       
 [73] rlang_0.4.10          pkgconfig_2.0.3       bitops_1.0-6         
 [76] evaluate_0.14         lattice_0.20-38       ROCR_1.0-7           
 [79] tensor_1.5            labeling_0.4.2        patchwork_1.1.1      
 [82] htmlwidgets_1.5.1     cowplot_1.1.1         tidyselect_1.1.0     
 [85] RcppAnnoy_0.0.18      plyr_1.8.6            magrittr_2.0.1       
 [88] R6_2.5.0              gplots_3.0.4          generics_0.1.0       
 [91] withr_2.4.2           pillar_1.4.7          mgcv_1.8-28          
 [94] fitdistrplus_1.0-14   survival_3.2-3        abind_1.4-5          
 [97] future.apply_1.6.0    crayon_1.3.4          KernSmooth_2.23-15   
[100] plotly_4.9.2.1        rmarkdown_2.3         locfit_1.5-9.4       
[103] grid_3.6.1            data.table_1.13.4     git2r_0.26.1         
[106] digest_0.6.27         xtable_1.8-4          httpuv_1.5.4         
[109] munsell_0.5.0         viridisLite_0.3.0    

sessionInfo()
R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
[1] C

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] ggplot2_3.3.3   harmony_1.0     Rcpp_1.0.6      purrr_0.3.4    
 [5] tibble_3.0.4    tidyr_1.1.0     dplyr_1.0.2     edgeR_3.28.1   
 [9] limma_3.42.2    Seurat_3.2.0    workflowr_1.6.2

loaded via a namespace (and not attached):
  [1] Rtsne_0.15            colorspace_2.0-0      deldir_0.1-28        
  [4] ellipsis_0.3.1        ggridges_0.5.2        rprojroot_2.0.2      
  [7] fs_1.4.2              spatstat.data_1.4-3   farver_2.0.3         
 [10] leiden_0.3.3          listenv_0.8.0         npsurv_0.4-0         
 [13] ggrepel_0.9.0         RSpectra_0.16-0       codetools_0.2-16     
 [16] splines_3.6.1         lsei_1.2-0            knitr_1.29           
 [19] polyclip_1.10-0       jsonlite_1.7.2        ica_1.0-2            
 [22] cluster_2.1.0         png_0.1-7             uwot_0.1.10          
 [25] shiny_1.5.0           sctransform_0.2.1     compiler_3.6.1       
 [28] httr_1.4.2            Matrix_1.2-18         fastmap_1.0.1        
 [31] lazyeval_0.2.2        later_1.1.0.1         htmltools_0.5.0      
 [34] tools_3.6.1           rsvd_1.0.3            igraph_1.2.6         
 [37] gtable_0.3.0          glue_1.4.2            RANN_2.6.1           
 [40] reshape2_1.4.4        rappdirs_0.3.3        spatstat_1.64-1      
 [43] vctrs_0.3.6           gdata_2.18.0          ape_5.4-1            
 [46] nlme_3.1-140          lmtest_0.9-37         xfun_0.16            
 [49] stringr_1.4.0         globals_0.12.5        mime_0.9             
 [52] miniUI_0.1.1.1        lifecycle_0.2.0       irlba_2.3.3          
 [55] gtools_3.8.2          goftest_1.2-2         future_1.18.0        
 [58] MASS_7.3-51.4         zoo_1.8-8             scales_1.1.1         
 [61] promises_1.1.1        spatstat.utils_1.17-0 parallel_3.6.1       
 [64] RColorBrewer_1.1-2    yaml_2.2.1            reticulate_1.20      
 [67] pbapply_1.4-2         gridExtra_2.3         rpart_4.1-15         
 [70] stringi_1.5.3         highr_0.8             caTools_1.18.0       
 [73] rlang_0.4.10          pkgconfig_2.0.3       bitops_1.0-6         
 [76] evaluate_0.14         lattice_0.20-38       ROCR_1.0-7           
 [79] tensor_1.5            labeling_0.4.2        patchwork_1.1.1      
 [82] htmlwidgets_1.5.1     cowplot_1.1.1         tidyselect_1.1.0     
 [85] RcppAnnoy_0.0.18      plyr_1.8.6            magrittr_2.0.1       
 [88] R6_2.5.0              gplots_3.0.4          generics_0.1.0       
 [91] withr_2.4.2           pillar_1.4.7          mgcv_1.8-28          
 [94] fitdistrplus_1.0-14   survival_3.2-3        abind_1.4-5          
 [97] future.apply_1.6.0    crayon_1.3.4          KernSmooth_2.23-15   
[100] plotly_4.9.2.1        rmarkdown_2.3         locfit_1.5-9.4       
[103] grid_3.6.1            data.table_1.13.4     git2r_0.26.1         
[106] digest_0.6.27         xtable_1.8-4          httpuv_1.5.4         
[109] munsell_0.5.0         viridisLite_0.3.0