Intratumoral heterogeneity in Glioblastoma
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> 1And 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 of tumour cell subclones (heatmap_subclones.png)
Heatmap of the Copy Number Alteration matrix with the clonal subpopulations found.
Clonal tree (CloneTree.png)
Clonal tree inferred from subsclone profiles.
Consensus plot (consensus.png)
Compact plot of the alterations present in each subpopulation.
OncoPrint-like plot (OncoHeat.png)
OncoPrint-like plot that highlighting specific alteration, shared alteration between subclones, or clonal alteration.
DE analysis in specific alterations (DEchr*_subclones.png)
Vulcano plot obtained from differential expression analysis of the genes belonging to the specific alterations found.
Pathway Analysis of subclones (pathwayAnalysis_subclones*.png)
REACTOME pathways activity obtained with GSEA for each subclone in contrast to the others for each subclone.
