Last updated: 2021-06-19

Checks: 4 3

Knit directory: MS_lesions/

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Rmd 8a4f558 Macnair 2021-06-15 Add analysis of lesion-specific genes
html 8a4f558 Macnair 2021-06-15 Add analysis of lesion-specific genes
Rmd 0564274 Macnair 2021-06-08 Add code for lesion-specific genes

Notes

With Tukey’s Honest Significant Difference test (HSD), the p-values are already adjusted within each gene + celltype. So to adjust them across the whole experiment, I adjusted within each individual comparison.

to do: - check WM results against muscat results (logFC; p-value)

Setup / definitions

Libraries

Helper functions

source('code/ms00_utils.R')
source('code/ms10_muscat_runs.R')
source('code/ms14_lesions.R')

Inputs

merged_f    = 'output/ms06_sccaf/conos_merged_dt_2021-03-18.csv'
labels_f    = 'data/byhand_markers/validation_markers_2021-05-31.csv'
n_cores     = 16

Outputs

save_dir    = 'output/ms14_lesions'
date_tag    = '2021-06-08'
if (!dir.exists(save_dir))
  dir.create(save_dir)
tukey_pat   = sprintf('%s/lesion_genes_tukey_%s_%s.txt.gz', 
  save_dir, '%s', date_tag)

# specify where soup and pseudobulk are located
pb_soup_f   = file.path(soup_dir, 'pb_soup_broad_maximum_2021-06-01.rds')
soup_cut    = 0.1

# specify what goes into muscat run
cluster_var = 'type_broad'
pb_f        = file.path(soup_dir, 'pb_sum_broad_2021-06-01.rds')
ranef_dt_f  = 'output/ms10_muscat/run09/muscat_ranef_dt_run09_2021-06-05.txt.gz'
formula_str = '~ lesion_type + sex + age_norm + pmi_cat'
subset_spec = list(matter = c('WM'))
random_var = 'patient_id'
filter      = 'both'
min_cells   = 10
p_cut       = 0.05

Load inputs

labels_dt   = .load_labels_dt(labels_f, cluster_var)
conos_dt    = merged_f %>% fread %>% 
  .[, .(cell_id, sample_id, conos = conos_merge)]
pb          = readRDS(pb_f) %>% .subset_pb(subset_spec)
  subsetting pb object
    restricting to samples that meet subset criteria
    updating factors to remove levels no longer observed
ranef_dt    = .load_ranef_dt(ranef_dt_f, labels_dt, pb)
myelin_ls   = get_myelin_ls()

Processing / calculations

calc_tukey_range_tests(tukey_pat, pb_f, subset_spec, 
  labels_f, cluster_var, formula_str, filter, min_cells, n_cores)
already done!
NULL
tukey_dt    = load_tukey_range_tests(tukey_pat, labels_f, cluster_var, 
  pb_f, pb_soup_f, min_cells, soup_cut)
loading tukey outputs
  annotating with soup
  adjusting p-values
tukey_ns    = calc_tukey_ns(tukey_dt, p_cut)
ranks_dt    = calc_tukey_ranks_dt(tukey_ns)
top_pats_dt = calc_top_patient_genes(ranef_dt)

Analysis

How many genes are characteristic for a particular lesion type?

print(plot_tukey_distns(tukey_ns))

Version Author Date
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Check suggested genes

for (l in levels(pb$lesion_type)) {
  cat('## Characteristic for ', l, ' lesions{.tabset}\n')
  for (cl in levels(labels_dt$cluster_id)) {
    cat('### ', cl, '\n')
    suppressWarnings(print(plot_best_gs(ranks_dt, l, cl, pb, top_n = 15)))
    cat('\n\n')
  }
}

Characteristic for WM lesions

OPCs / COPs

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Oligodendrocytes

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Astrocytes

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Microglia

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Excitatory neurons

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Inhibitory neurons

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Endothelial cells

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Pericytes

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Immune

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Characteristic for NAWM lesions

OPCs / COPs

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Oligodendrocytes

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Astrocytes

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Microglia

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Excitatory neurons

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Inhibitory neurons

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Endothelial cells

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Pericytes

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Immune

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Characteristic for AL lesions

OPCs / COPs

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Oligodendrocytes

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Astrocytes

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Microglia

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Excitatory neurons

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Inhibitory neurons

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Endothelial cells

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Pericytes

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Immune

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Characteristic for CAL lesions

OPCs / COPs

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Oligodendrocytes

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Astrocytes

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Microglia

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Excitatory neurons

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8a4f558 Macnair 2021-06-15

Inhibitory neurons

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8a4f558 Macnair 2021-06-15

Endothelial cells

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8a4f558 Macnair 2021-06-15

Pericytes

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Immune

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Characteristic for CIL lesions

OPCs / COPs

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Oligodendrocytes

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Astrocytes

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Microglia

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Excitatory neurons

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8a4f558 Macnair 2021-06-15

Inhibitory neurons

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8a4f558 Macnair 2021-06-15

Endothelial cells

Version Author Date
8a4f558 Macnair 2021-06-15

Pericytes

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8a4f558 Macnair 2021-06-15

Immune

Version Author Date
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Characteristic for RL lesions

OPCs / COPs

Version Author Date
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Oligodendrocytes

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Astrocytes

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8a4f558 Macnair 2021-06-15

Microglia

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Excitatory neurons

Version Author Date
8a4f558 Macnair 2021-06-15

Inhibitory neurons

Version Author Date
8a4f558 Macnair 2021-06-15

Endothelial cells

Version Author Date
8a4f558 Macnair 2021-06-15

Pericytes

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8a4f558 Macnair 2021-06-15

Immune

Version Author Date
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logCPM plots of genes varying most between patients

for (cl in levels(labels_dt$cluster_id)) {
  cat('### ', cl, '\n')
  suppressWarnings(print(plot_top_pats(top_pats_dt, cl, pb, top_n = 15)))
  cat('\n\n')
}

OPCs / COPs

Oligodendrocytes

Astrocytes

Microglia

Excitatory neurons

Inhibitory neurons

Endothelial cells

Pericytes

Immune

logCPM plots of myelin genes

for (n in names(myelin_ls)) {
  cat('### ', n, '\n')
  suppressWarnings(print(plot_myelin_genes(myelin_ls[[n]], 
    'Oligodendrocytes', pb)))
  cat('\n\n')
}

myelin_maintenance

myelin_assembly

Outputs

devtools::session_info()
- Session info ---------------------------------------------------------------
 setting  value                       
 version  R version 4.0.3 (2020-10-10)
 os       CentOS Linux 7 (Core)       
 system   x86_64, linux-gnu           
 ui       X11                         
 language (EN)                        
 collate  en_US.UTF-8                 
 ctype    C                           
 tz       Europe/Zurich               
 date     2021-06-10                  

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 scater               * 1.18.6     2021-02-26 [1]
 sctransform            0.3.2      2020-12-16 [2]
 scuttle                1.0.4      2020-12-17 [1]
 seriation            * 1.2-9      2020-10-01 [1]
 sessioninfo            1.1.1      2018-11-05 [1]
 shape                  1.4.5      2020-09-13 [2]
 SingleCellExperiment * 1.12.0     2020-10-27 [1]
 snakecase              0.11.0     2019-05-25 [1]
 sparseMatrixStats      1.2.1      2021-02-02 [1]
 stringi                1.6.2      2021-05-17 [2]
 stringr              * 1.4.0      2019-02-10 [2]
 SummarizedExperiment * 1.20.0     2020-10-27 [1]
 survival             * 3.2-11     2021-04-26 [2]
 testthat               3.0.2      2021-02-14 [2]
 TH.data              * 1.0-10     2019-01-21 [1]
 tibble               * 3.1.2      2021-05-16 [2]
 tictoc               * 1.0.1      2021-04-19 [1]
 tidyr                * 1.1.3      2021-03-03 [2]
 tidyselect             1.1.1      2021-04-30 [2]
 tidyverse            * 1.3.1      2021-04-15 [2]
 TMB                    1.7.20     2021-04-08 [1]
 TSP                    1.1-10     2020-04-17 [1]
 UpSetR               * 1.4.0      2019-05-22 [1]
 usethis                2.0.1      2021-02-10 [1]
 utf8                   1.2.1      2021-03-12 [2]
 variancePartition      1.20.0     2020-10-27 [1]
 vctrs                  0.3.8      2021-04-29 [2]
 vipor                  0.4.5      2017-03-22 [1]
 viridis              * 0.6.1      2021-05-11 [1]
 viridisLite          * 0.4.0      2021-04-13 [2]
 whisker                0.4        2019-08-28 [1]
 withr                  2.4.2      2021-04-18 [2]
 workflowr            * 1.6.2      2020-04-30 [1]
 writexl              * 1.4.0      2021-04-20 [1]
 xfun                   0.23       2021-05-15 [1]
 XML                    3.99-0.6   2021-03-16 [1]
 xml2                   1.3.2      2020-04-23 [2]
 xtable                 1.8-4      2019-04-21 [2]
 XVector                0.30.0     2020-10-27 [1]
 yaml                   2.2.1      2020-02-01 [2]
 zlibbioc               1.36.0     2020-10-27 [1]
 zoo                    1.8-9      2021-03-09 [2]
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[1] /pstore/home/macnairw/lib/conda_r3.12
[2] /pstore/home/macnairw/.conda/envs/r_4.0.3/lib/R/library

sessionInfo()
R version 4.0.3 (2020-10-10)
Platform: x86_64-conda-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

Matrix products: default
BLAS/LAPACK: /pstore/home/macnairw/.conda/envs/r_4.0.3/lib/libopenblasp-r0.3.12.so

locale:
 [1] LC_CTYPE=C                 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] grid      parallel  stats4    stats     graphics  grDevices utils    
 [8] datasets  methods   base     

other attached packages:
 [1] ggbeeswarm_0.6.0            multcomp_1.4-17            
 [3] TH.data_1.0-10              MASS_7.3-54                
 [5] survival_3.2-11             mvtnorm_1.1-1              
 [7] rmarkdown_2.8               ggrepel_0.9.1              
 [9] patchwork_1.1.1             writexl_1.4.0              
[11] ComplexHeatmap_2.6.2        fgsea_1.16.0               
[13] tictoc_1.0.1                edgeR_3.32.1               
[15] limma_3.46.0                reshape2_1.4.4             
[17] scater_1.18.6               Matrix.utils_0.9.8         
[19] Matrix_1.3-3                SingleCellExperiment_1.12.0
[21] SummarizedExperiment_1.20.0 Biobase_2.50.0             
[23] MatrixGenerics_1.2.1        matrixStats_0.58.0         
[25] seriation_1.2-9             UpSetR_1.4.0               
[27] BiocParallel_1.24.1         muscat_1.5.1               
[29] dplyr_1.0.6                 purrr_0.3.4                
[31] readr_1.4.0                 tidyr_1.1.3                
[33] tibble_3.1.2                tidyverse_1.3.1            
[35] rtracklayer_1.50.0          GenomicRanges_1.42.0       
[37] GenomeInfoDb_1.26.7         IRanges_2.24.1             
[39] S4Vectors_0.28.1            BiocGenerics_0.36.1        
[41] forcats_0.5.1               ggplot2_3.3.3              
[43] scales_1.1.1                viridis_0.6.1              
[45] viridisLite_0.4.0           assertthat_0.2.1           
[47] stringr_1.4.0               data.table_1.14.0          
[49] magrittr_2.0.1              circlize_0.4.12            
[51] RColorBrewer_1.1-2          BiocStyle_2.18.1           
[53] colorout_1.2-2              workflowr_1.6.2            

loaded via a namespace (and not attached):
  [1] R.methodsS3_1.8.1         bit64_4.0.5              
  [3] knitr_1.33                R.utils_2.10.1           
  [5] irlba_2.3.3               DelayedArray_0.16.3      
  [7] RCurl_1.98-1.3            doParallel_1.0.16        
  [9] generics_0.1.0            callr_3.7.0              
 [11] usethis_2.0.1             RSQLite_2.2.7            
 [13] future_1.21.0             bit_4.0.4                
 [15] xml2_1.3.2                lubridate_1.7.10         
 [17] httpuv_1.6.1              xfun_0.23                
 [19] hms_1.1.0                 jquerylib_0.1.4          
 [21] evaluate_0.14             promises_1.2.0.1         
 [23] TSP_1.1-10                fansi_0.4.2              
 [25] progress_1.2.2            caTools_1.18.2           
 [27] dbplyr_2.1.1              readxl_1.3.1             
 [29] DBI_1.1.1                 geneplotter_1.68.0       
 [31] ellipsis_0.3.2            backports_1.2.1          
 [33] annotate_1.68.0           sparseMatrixStats_1.2.1  
 [35] vctrs_0.3.8               remotes_2.3.0            
 [37] Cairo_1.5-12.2            cachem_1.0.5             
 [39] withr_2.4.2               grr_0.9.5                
 [41] sctransform_0.3.2         GenomicAlignments_1.26.0 
 [43] prettyunits_1.1.1         cluster_2.1.2            
 [45] crayon_1.4.1              genefilter_1.72.1        
 [47] labeling_0.4.2            pkgconfig_2.0.3          
 [49] pkgload_1.2.1             nlme_3.1-152             
 [51] vipor_0.4.5               devtools_2.4.1           
 [53] blme_1.0-5                rlang_0.4.11             
 [55] globals_0.14.0            lifecycle_1.0.0          
 [57] sandwich_3.0-1            registry_0.5-1           
 [59] modelr_0.1.8              rsvd_1.0.5               
 [61] cellranger_1.1.0          rprojroot_2.0.2          
 [63] boot_1.3-28               zoo_1.8-9                
 [65] reprex_2.0.0              beeswarm_0.3.1           
 [67] processx_3.5.2            whisker_0.4              
 [69] GlobalOptions_0.1.2       png_0.1-7                
 [71] rjson_0.2.20              bitops_1.0-7             
 [73] R.oo_1.24.0               KernSmooth_2.23-20       
 [75] Biostrings_2.58.0         blob_1.2.1               
 [77] DelayedMatrixStats_1.12.3 shape_1.4.5              
 [79] parallelly_1.25.0         beachmat_2.6.4           
 [81] memoise_2.0.0             plyr_1.8.6               
 [83] gplots_3.1.1              zlibbioc_1.36.0          
 [85] compiler_4.0.3            clue_0.3-59              
 [87] lme4_1.1-27               DESeq2_1.30.1            
 [89] snakecase_0.11.0          Rsamtools_2.6.0          
 [91] cli_2.5.0                 XVector_0.30.0           
 [93] lmerTest_3.1-3            listenv_0.8.0            
 [95] ps_1.6.0                  TMB_1.7.20               
 [97] tidyselect_1.1.1          stringi_1.6.2            
 [99] highr_0.9                 yaml_2.2.1               
[101] BiocSingular_1.6.0        locfit_1.5-9.4           
[103] sass_0.4.0                fastmatch_1.1-0          
[105] tools_4.0.3               future.apply_1.7.0       
[107] rstudioapi_0.13           foreach_1.5.1            
[109] git2r_0.28.0              janitor_2.1.0            
[111] gridExtra_2.3             farver_2.1.0             
[113] digest_0.6.27             BiocManager_1.30.15      
[115] Rcpp_1.0.6                broom_0.7.6              
[117] scuttle_1.0.4             later_1.2.0              
[119] httr_1.4.2                AnnotationDbi_1.52.0     
[121] colorspace_2.0-1          rvest_1.0.0              
[123] XML_3.99-0.6              fs_1.5.0                 
[125] splines_4.0.3             sessioninfo_1.1.1        
[127] xtable_1.8-4              jsonlite_1.7.2           
[129] nloptr_1.2.2.2            testthat_3.0.2           
[131] R6_2.5.0                  pillar_1.6.1             
[133] htmltools_0.5.1.1         glue_1.4.2               
[135] fastmap_1.1.0             minqa_1.2.4              
[137] BiocNeighbors_1.8.2       codetools_0.2-18         
[139] pkgbuild_1.2.0            utf8_1.2.1               
[141] lattice_0.20-44           bslib_0.2.5              
[143] numDeriv_2016.8-1.1       pbkrtest_0.5.1           
[145] colorRamps_2.3            gtools_3.8.2             
[147] glmmTMB_1.0.2.1           desc_1.3.0               
[149] munsell_0.5.0             GetoptLong_1.0.5         
[151] GenomeInfoDbData_1.2.4    iterators_1.0.13         
[153] variancePartition_1.20.0  haven_2.4.1              
[155] gtable_0.3.0