Last updated: 2018-11-05

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      Modified:   analysis/10x-180504-general-analysis.Rmd
      Modified:   code/10x-180504-preprocessing.R
      Modified:   plots/180504_pca_tsne.pdf
      Modified:   plots/supplementary_figures/sfig_180504_pcelbow.pdf
      Modified:   plots/supplementary_figures/sfig_180504_qcplots.pdf
  
  

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Plots of 10x-180504 before quality control.

library(Seurat)

Loading required package: ggplot2

Loading required package: cowplot

Attaching package: 'cowplot'

The following object is masked from 'package:ggplot2':

    ggsave

Loading required package: Matrix

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

all10x.beforeqc <- readRDS('output/10x-180504-beforeQC')
all10x <- readRDS('output/10x-180504')

pm <- VlnPlot(all10x.beforeqc, features.plot='percent.mito', group.by='sample_name', point.size.use = -1, do.return=T, x.lab.rot=T, size.x.use=8) + geom_hline(yintercept=0.08, linetype="dashed", color = "red")
pm

numi <- VlnPlot(all10x.beforeqc, features.plot='nUMI', group.by='sample_name', point.size.use = -1, do.return=T, x.lab.rot=T, size.x.use=8) + geom_hline(yintercept=110000, linetype="dashed", color = "red")
numi

ngene <- VlnPlot(all10x.beforeqc, features.plot='nGene', group.by='sample_name', point.size.use = -1, do.return=T, x.lab.rot=T, size.x.use=8) + geom_hline(yintercept=200, linetype="dashed", color = "red") + geom_hline(yintercept=9000, linetype="dashed", color = "red")
ngene

Warning: Removed 1 rows containing missing values (geom_hline).

GenePlot(all10x.beforeqc, 'nUMI', 'nGene', cex.use = 0.2, do.return=T)

Warning in plot.window(...): "do.return" is not a graphical parameter

Warning in plot.xy(xy, type, ...): "do.return" is not a graphical parameter

Warning in axis(side = side, at = at, labels = labels, ...): "do.return" is
not a graphical parameter

Warning in axis(side = side, at = at, labels = labels, ...): "do.return" is
not a graphical parameter

Warning in box(...): "do.return" is not a graphical parameter

Warning in title(...): "do.return" is not a graphical parameter

Number of cells before and after QC

before <- all10x.beforeqc@meta.data %>% count(sample_name)
after <- all10x@meta.data %>% count(sample_name)
ncells <- merge(before, after, by='sample_name', suffixes=c('.before', '.after'))
ncells

   sample_name n.before n.after
1       Peri_1     3973    3934
2       Peri_2     2889    2852
3       Peri_3     1346    1273
4       Subq_1     3087    3007
5       Subq_2     2071    2002
6       Subq_3     8661    8619
7       Subq_4     5022    4885
8      Supra_1     3056    3047
9      Supra_2     3664    3570
10     Supra_3     1035     880
11     Supra_4     3014    2785
12     Visce_1     4624    4539
13     Visce_2     4956    4893
14     Visce_3    10121   10085

write.table(ncells, file='tables/10x-180504-nCells.txt', sep='\t', row.names = F, quote=F)

sfig <- plot_grid(
  pm,
  numi,
  ngene,
  labels="auto", nrow=1
)

Warning: Removed 1 rows containing missing values (geom_hline).

save_plot("plots/supplementary_figures/sfig_180504_qcplots.pdf", sfig, base_width=12, base_height=3)
sfig

sessionInfo()

R version 3.4.3 (2017-11-30)
Platform: x86_64-redhat-linux-gnu (64-bit)
Running under: Red Hat Enterprise Linux

Matrix products: default
BLAS/LAPACK: /usr/lib64/R/lib/libRblas.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

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

other attached packages:
[1] dplyr_0.7.6   Seurat_2.3.4  Matrix_1.2-14 cowplot_0.9.3 ggplot2_3.0.0

loaded via a namespace (and not attached):
  [1] Rtsne_0.13          colorspace_1.3-2    class_7.3-14       
  [4] modeltools_0.2-22   ggridges_0.5.0      mclust_5.4.1       
  [7] rprojroot_1.3-2     htmlTable_1.12      base64enc_0.1-3    
 [10] rstudioapi_0.7      proxy_0.4-22        flexmix_2.3-14     
 [13] bit64_0.9-7         mvtnorm_1.0-8       codetools_0.2-15   
 [16] splines_3.4.3       R.methodsS3_1.7.1   robustbase_0.93-2  
 [19] knitr_1.20          Formula_1.2-3       jsonlite_1.5       
 [22] workflowr_1.1.1     ica_1.0-2           cluster_2.0.7-1    
 [25] kernlab_0.9-27      png_0.1-7           R.oo_1.22.0        
 [28] compiler_3.4.3      httr_1.3.1          backports_1.1.2    
 [31] assertthat_0.2.0    lazyeval_0.2.1      lars_1.2           
 [34] acepack_1.4.1       htmltools_0.3.6     tools_3.4.3        
 [37] bindrcpp_0.2.2      igraph_1.2.2        gtable_0.2.0       
 [40] glue_1.3.0          RANN_2.6            reshape2_1.4.3     
 [43] Rcpp_0.12.18        trimcluster_0.1-2.1 gdata_2.18.0       
 [46] ape_5.1             nlme_3.1-137        iterators_1.0.10   
 [49] fpc_2.1-11.1        gbRd_0.4-11         lmtest_0.9-36      
 [52] stringr_1.3.1       irlba_2.3.2         gtools_3.8.1       
 [55] DEoptimR_1.0-8      MASS_7.3-50         zoo_1.8-3          
 [58] scales_1.0.0        doSNOW_1.0.16       parallel_3.4.3     
 [61] RColorBrewer_1.1-2  yaml_2.2.0          reticulate_1.10    
 [64] pbapply_1.3-4       gridExtra_2.3       rpart_4.1-13       
 [67] segmented_0.5-3.0   latticeExtra_0.6-28 stringi_1.2.4      
 [70] foreach_1.4.4       checkmate_1.8.5     caTools_1.17.1.1   
 [73] bibtex_0.4.2        Rdpack_0.9-0        SDMTools_1.1-221   
 [76] rlang_0.2.2         pkgconfig_2.0.2     dtw_1.20-1         
 [79] prabclus_2.2-6      bitops_1.0-6        evaluate_0.11      
 [82] lattice_0.20-35     ROCR_1.0-7          purrr_0.2.5        
 [85] bindr_0.1.1         labeling_0.3        htmlwidgets_1.2    
 [88] bit_1.1-14          tidyselect_0.2.4    plyr_1.8.4         
 [91] magrittr_1.5        R6_2.2.2            snow_0.4-2         
 [94] gplots_3.0.1        Hmisc_4.1-1         pillar_1.3.0       
 [97] whisker_0.3-2       foreign_0.8-70      withr_2.1.2        
[100] fitdistrplus_1.0-9  mixtools_1.1.0      survival_2.42-6    
[103] nnet_7.3-12         tsne_0.1-3          tibble_1.4.2       
[106] crayon_1.3.4        hdf5r_1.0.0         KernSmooth_2.23-15 
[109] rmarkdown_1.10      grid_3.4.3          data.table_1.11.4  
[112] git2r_0.23.0        metap_1.0           digest_0.6.15      
[115] diptest_0.75-7      tidyr_0.8.1         R.utils_2.7.0      
[118] stats4_3.4.3        munsell_0.5.0      

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