Load data

We load the scRNA data of MGH106 sample from the public dataset of Gliobastoma (GSE131928)

load(url("https://www.dropbox.com/s/b9udpvhnc2ez9pc/MGH106_data.RData?raw=1"))

Run pipeline

We run the pipeline that performs the classification of malignant and non-malignant cells of the MGH106 sample using 20 cores and requesting analysis of possible subpopulations.

library(SCEVAN)
results <- SCEVAN::pipelineCNA(count_mtx, sample = "MGH106", par_cores = 20, SUBCLONES = TRUE, plotTree = TRUE)
## [1] " raw data - genes: 23686 cells: 200"
## [1] "1) Filter: cells > 200 genes"
## [1] "2) Filter: genes > 10% of cells"
## [1] "12304 genes past filtering"
## [1] "3) Annotations gene coordinates"
## [1] "found 30 confident non malignant cells"
## [1] "11068 genes annotated"
## [1] "4) Filter: genes involved in the cell cycle"
## [1] "10566 genes past filtering "
## [1] "5)  Filter: cells > 5genes per chromosome "
## [1] "6) Log Freeman Turkey transformation"
## [1] "A total of 200 cells, 10566 genes after preprocessing"
## [1] "7) Measuring baselines (confident normal cells)"
## [1] "8) Smoothing data"
## [1] "9) Segmentation (VegaMC)"
## [1] "10) Adjust baseline"
## [1] "11) plot heatmap"
## [1] "found 136 tumor cells"
## [1] "time classify tumor cells:  25.8686459064484"
## [1] "found 3 subclones"
## percentage_cells_subsclone_1 percentage_cells_subsclone_2 percentage_cells_subsclone_3 
##                    0.3897059                    0.4191176                    0.1911765 
## [1] "Segmentation of subclone :  1"
## [1] "Segmentation of subclone :  2"
## [1] "Segmentation of subclone :  3"
## $MGH106_subclone1
##     Chr     Start       End Alteration segm.mean
## 16    1 155262868 156430701          2  0.191559
## 36    2 132671799 162371595         -2 -0.220405
## 76    5 132866560 141636870          2  0.147716
## 89    6 129576132 148552050         -2 -0.177527
## 106   8  17643795  28402701          2  0.165937
## 123   9 124777158 128204383         -2 -0.142754
## 158  12  63142759  76853696         -2 -0.147801
## 207  17  29625938  36543435         -2 -0.175544
## 212  17  48830988  51171747          2  0.122247
## 216  17  76389451  78782297         -2 -0.159493
## 232  20   3846799  18316996          2  0.149706
## 247  22  16601887  18914238          2  0.099859
## 120   9  92210207  99970341         -1 -0.063417
## 193  16   2513870  11797267         -1 -0.092448
## 227  19  27684580  34471251          1  0.156300
## 
## $MGH106_subclone2
##     Chr     Start       End Alteration segm.mean
## 8     1 151281618 157045370          2  0.188821
## 10    1 206571292 212816626         -2 -0.332493
## 13    2    217730  28870301          2  0.099843
## 20    2  86440647 166149214         -2 -0.219312
## 41    4  26576437  39639090         -2 -0.244094
## 49    4 158672125 174284264          2  0.257183
## 59    5 115124762 139482935          2  0.190356
## 96    8    213186  28755599          2  0.190994
## 100   8  73991352  92103286          2  0.207449
## 128  11  46846412  58578220          2  0.207355
## 163  13 102844844 110870251          2  0.279267
## 190  16  53701692  65126112          2  0.460633
## 199  17  30629680  39730426         -2 -0.264341
## 201  17  40818117  55421992          2  0.275907
## 227  20    270863  34955817          2  0.164324
## 4     1  43164175  78646145          1  0.131879
## 52    5   6599239  32604079          1  0.157283
## 73    6  87470623 107660167         -1 -0.104160
## 75    6 131135666 163315492         -1 -0.122239
## 98    8  38988808  58659844          1  0.153508
## 106   9  86264546  96014571         -1 -0.155870
## 187  16   2239395  31917357         -1 -0.101952
## 220  19  19645198  29617237         -1 -0.124974
## 221  19  29698886  36528660          1  0.157899
## 
## $MGH106_subclone3
##     Chr     Start       End Alteration segm.mean
## 2     1   6634168  11725857          2  0.203431
## 41    3 113532296 138633376          2  0.168362
## 45    3 158644278 194498364          2  0.280193
## 62    5 134928039 149369653          2  0.257570
## 73    6 109390215 125980244          2  0.161127
## 74    6 125986430 160113507         -2 -0.215176
## 103   9 112217716 117716871         -2 -0.208159
## 147  14  18967434  23299447          2  0.166348
## 184  17  29589769  48929056         -2 -0.266468
## 211  20   1442162  43550950          2  0.236827
## 219  21  25716503  33643926          2  0.172607
## 231  22  42617840  46357340         -2 -0.127763
## 5     1  29192873  36156053          1  0.127030
## 8     1  77088990  93278730         -1 -0.137756
## 13    1 155320896 160358952          1  0.206748
## 24    2 130074030 175168382         -1 -0.120301
## 37    3  50329782  52239260         -1 -0.042777
## 51    4  25312785  46475230         -1 -0.176821
## 52    4  47450796  56464579          1  0.143567
## 87    8  40153455  63039171          1  0.135214
## 117  11    167784   6481479         -1 -0.088935
## 123  11 111602391 134411918          1  0.119784
## 135  12  57095408  70637440         -1 -0.165177
## 141  12 120533480 123244014         -1 -0.104119
## 171  16   2223566   4242080         -1 -0.120347
## 230  22  40370591  42640601          1  0.071973
## 
## $MGH106_clone
##     Chr     Start       End Alteration segm.mean
## 2     1  25818640  28643085         -2 -0.214814
## 40    2 190189735 199471266         -2 -0.233109
## 44    2 215996329 232860575         -2 -0.201109
## 47    3  14402576  47413441         -2 -0.238216
## 49    3  47562239  50322906          2  0.216302
## 59    5  42423777  55922853         -2 -0.291359
## 80    5 157029413 172771195         -2 -0.311226
## 68    6  13621498  42452051         -2 -0.249160
## 92    7    192969 159144957          2  0.197698
## 115   9     14521  36124451         -2 -0.199105
## 128  10    134465 133421307         -2 -0.344739
## 133  11  66848233  68036644         -2 -0.155899
## 153  12   6988259  16610594         -2 -0.221581
## 161  12 109900264 111817529          2  0.104997
## 165  13  18816718  52194465         -2 -0.168552
## 201  17    212389  12143830         -2 -0.212809
## 223  19     60951  17405648          2  0.216058
## 238  20  50081124  58309439         -2 -0.161519
## 253  22  31618491  39973721         -2 -0.038629
## 
## $MGH106_shareSubclone
##                   Chr     Start       End Alteration segm.mean sh_sub Mean
## MGH106_share1.4     1  23964804  27336400         -2 -0.246198    1-2    0
## MGH106_share3.4     1  25818640  28643085         -2 -0.211230    3-2    0
## MGH106_share1.19    1 185296388 212816626         -2 -0.176747    1-3    0
## MGH106_share3.15    1 206571292 212816626         -2 -0.220886    3-1    0
## MGH106_share1.32    2  85318020 112589275         -2 -0.217311    1-3    0
## MGH106_share3.27    2 215996329 223602503          2  0.198019    3-2    0
## MGH106_share1.43    2 216672105 223198399          2  0.189197    1-2    0
## MGH106_share1.70    5  37379212  56896152         -2 -0.251641    1-3    0
## MGH106_share2.54    5  42423777  55922853         -2 -0.295466    2-3    0
## MGH106_share1.84    6  13621498  30327150         -2 -0.114650    1-3    0
## MGH106_share2.68    6  13621498  42452051         -2 -0.103184    2-3    0
## MGH106_share1.91    6 163414000 170584692          2  0.138883    1-2    0
## MGH106_share1.113   8 132685007 143603224         -1 -0.169084    1-2    0
## MGH106_share1.146  11  66848233  67508649         -2 -0.235590    1-2    0
## MGH106_share3.120  11  66848233  68036644         -2 -0.184764    3-2    0
## MGH106_share1.150  11 116748170 119003446         -2 -0.171403    1-2    0
## MGH106_share2.153  12 109116595 111817529          2  0.189422    2-1    0
## MGH106_share1.170  13 106489731 114327328          2  0.138539    1-3    0
## MGH106_share1.182  14  88005124  93116320         -2 -0.153034    1-3    0
## MGH106_share1.184  14 102317377 104768494          2  0.171552    1-3    0
## MGH106_share2.175  15  34851782  43371025         -1 -0.159688    2-3    0
## MGH106_share2.182  15  75353611  78299794          1  0.108603    2-3    0
## MGH106_share2.186  16     46407   2209416          2  0.173651    2-3    0
## MGH106_share1.195  16  28591943  30446181         -2 -0.124534    1-3    0
## MGH106_share1.197  16  55389700  57447528          2  0.284093    1-3    0
## MGH106_share2.197  17  28335602  28903071          2  0.198159    2-3    0
## MGH106_share2.212  19     60951  11784078          2  0.223597    2-1    0
## MGH106_share3.199  19  29698886  36528660          2  0.257608    3-1    0
## MGH106_share2.223  19  44537058  54102692         -2 -0.188791    2-3    0
## MGH106_share1.241  21  17901263  32393026          2  0.172096    1-2    0
## MGH106_share1.246  21  45263928  46665124          2  0.103196    1-3    0
## MGH106_share2.242  22  36189124  39532855         -2 -0.210030    2-1    0
## [1] "time subclones:  4.65857791105906"

Output

The pipeline returns in results a data frame containing for each cell its classification (tumor/normal), whether it has been used as a normal confident cell, and to which clonal subpopulation it belongs.

head(results)
##               class confidentNormal subclone
## MGH106-P7-E10 tumor            <NA>        1
## MGH106-P2-B01 tumor            <NA>        1
## MGH106-P2-B12 tumor            <NA>        1
## MGH106-P2-H07 tumor            <NA>        2
## MGH106-P2-E09 tumor            <NA>        1
## MGH106-P2-E04 tumor            <NA>        1

And creates an output folder containing all segmentation output files and all the plots generated by the analysis:

list.files(path = "./output", pattern = "MGH106")
##  [1] " MGH106 vega_output"                              " MGH106_SubcloneDiffAnalysis.RData"               " MGH106onlytumor vega_output"                    
##  [4] "MGH106_Clonal_CN.seg"                             "MGH106_CNAmtx.RData"                              "MGH106_CNAmtxSubclones.RData"                    
##  [7] "MGH106_count_mtx_annot.RData"                     "MGH106_subclone1_CN.seg"                          "MGH106_subclone2_CN.seg"                         
## [10] "MGH106_subclone3_CN.seg"                          "MGH106- DEchr12:48926474:50821122_subclones.png"  "MGH106- DEchr3:112585577:198043720_subclones.png"
## [13] "MGH106- DEchr3:409417:14489349_subclones.png"     "MGH106-DEchr1-155320896-160358952_subclones.png"  "MGH106-DEchr1-185045364-212899363_subclones.png" 
## [16] "MGH106-DEchr1-29192873-36180849_subclones.png"    "MGH106-DEchr1-74705482-93278730_subclones.png"    "MGH106-DEchr11-111602391-134411918_subclones.png"
## [19] "MGH106-DEchr11-86244544-123063230_subclones.png"  "MGH106-DEchr12_subclones.png"                     "MGH106-DEchr12-109088188-120904371_subclones.png"
## [22] "MGH106-DEchr16-2239395-31917357_subclones.png"    "MGH106-DEchr16-2513870-11797267_subclones.png"    "MGH106-DEchr17-40309192-55325065_subclones.png"  
## [25] "MGH106-DEchr17-62423867-79049788_subclones.png"   "MGH106-DEchr17-76389451-78782297_subclones.png"   "MGH106-DEchr19-44165073-57578927_subclones.png"  
## [28] "MGH106-DEchr19-47870466-54102692_subclones.png"   "MGH106-DEchr2-130074030-175168382_subclones.png"  "MGH106-DEchr21-25585656-46665124_subclones.png"  
## [31] "MGH106-DEchr21-25716503-33643926_subclones.png"   "MGH106-DEchr21-28872191-33643926_subclones.png"   "MGH106-DEchr3_subclones.png"                     
## [34] "MGH106-DEchr3-112561709-198043720_subclones.png"  "MGH106-DEchr3-158644278-194498364_subclones.png"  "MGH106-DEchr4-108810721-129093607_subclones.png" 
## [37] "MGH106-DEchr4-154626945-184221230_subclones.png"  "MGH106-DEchr4-158672125-174284264_subclones.png"  "MGH106-DEchr4-64275257-77952790_subclones.png"   
## [40] "MGH106-DEchr5-108747822-149944793_subclones.png"  "MGH106-DEchr5-115124762-139482935_subclones.png"  "MGH106-DEchr5-134928039-149369653_subclones.png" 
## [43] "MGH106-DEchr5-98853985-140549578_subclones.png"   "MGH106-DEchr6-109390215-125980244_subclones.png"  "MGH106-DEchr6-125986430-160113507_subclones.png" 
## [46] "MGH106-DEchr6-291630-36232790_subclones.png"      "MGH106-DEchr8-126847055-143733801_subclones.png"  "MGH106-DEchr8-213186-28755599_subclones.png"     
## [49] "MGH106-DEchr8-73991352-92103286_subclones.png"    "MGH106-DEchr8-75407914-92103286_subclones.png"    "MGH106CloneTree.png"                             
## [52] "MGH106consensus.png"                              "MGH106heatmap_subclones.png"                      "MGH106heatmap.png"                               
## [55] "MGH106OncoHeat.png"                               "MGH106OncoHeat2.png"                              "MGH106onlytumorheatmap.png"                      
## [58] "MGH106pathwayAnalysis_subclones1.png"             "MGH106pathwayAnalysis_subclones2.png"             "MGH106pathwayAnalysis_subclones3.png"            
## [61] "MGH106plotCNline.png"                             "MGH106tsne_CNA.png"                               "MGH106tsne_scRNA.png"

Heatmap classification of tumor cells (heatmap.png)

Heatmap of the Copy Number Alteration matrix with classification of non-malignant and malignant cells.

heatmap

Heatmap of tumour cell subclones (heatmap_subclones.png)

Heatmap of the Copy Number Alteration matrix with the clonal subpopulations found.

heatmap

Clonal tree (CloneTree.png)

Clonal tree inferred from subsclone profiles.

heatmap

Consensus plot (consensus.png)

Compact plot of the alterations present in each subpopulation.

heatmap

OncoPrint-like plot (OncoHeat.png)

OncoPrint-like plot that highlighting specific alteration, shared alteration between subclones, or clonal alteration.

heatmap

DE analysis in specific alterations (DEchr*_subclones.png)

Vulcano plot obtained from differential expression analysis of the genes belonging to the specific alterations found.

heatmap

heatmap

Pathway Analysis of subclones (pathwayAnalysis_subclones*.png)

REACTOME pathways activity obtained with GSEA for each subclone in contrast to the others for each subclone.

heatmap heatmap heatmap