DLPFC
Last updated: 2024-07-14
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Knit directory: KODAMA-Analysis/
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| Rmd | 0051854 | Stefano Cacciatore | 2024-07-14 | Start my new project |
We will analyze all 12 slides
library("nnSVG")
library("scater")
library("scran")
library("scry")
library("SPARK")
library("harmony")
library("Seurat")
library("spatialLIBD")
library("KODAMA")
library("KODAMAextra")
sample_names=c("151507",
"151508",
"151509",
"151510",
"151669",
"151670",
"151671",
"151672",
"151673",
"151674",
"151675",
"151676")
subject_names= c("Br5292","Br5595", "Br8100")
#spe <- fetch_data(type = 'spe',destdir="../Temporary")
load("../DLFPC.RData")
metaData = SingleCellExperiment::colData(spe)
expr = SingleCellExperiment::counts(spe)
sample_names <- paste0("sample_", unique(colData(spe)$sample_id))
sample_names <- unique(colData(spe)$sample_id)
dim(spe)[1] 33538 47681identify mitochondrial genes
is_mito <- grepl("(^MT-)|(^mt-)", rowData(spe)$gene_name)
table(is_mito)is_mito
FALSE TRUE
33525 13 calculate per-spot QC metrics and select the QC threshoulds
spe <- addPerCellQC(spe, subsets = list(mito = is_mito))
# select QC thresholds
qc_lib_size <- colData(spe)$sum < 500
qc_detected <- colData(spe)$detected < 250
qc_mito <- colData(spe)$subsets_mito_percent > 30
qc_cell_count <- colData(spe)$cell_count > 12Spot to discard and filter low-quality
discard <- qc_lib_size | qc_detected | qc_mito | qc_cell_count
table(discard)discard
FALSE TRUE
46653 1028 colData(spe)$discard <- discard
spe <- spe[, !colData(spe)$discard]
dim(spe)[1] 33538 46653Horizontalization
# readjust xy
xy=spatialCoords(spe)
samples=unique(colData(spe)$sample_id)
for(j in 1:length(samples)){
sel=samples[j]==colData(spe)$sample_id
xy[sel,1]=spatialCoords(spe)[sel,1]+12000*(j-1)
}
spatialCoords(spe)=xyGene filtering
spe <- filter_genes(
spe,
filter_genes_ncounts = 2, #ncounts
filter_genes_pcspots = 0.5,
filter_mito = TRUE
)
dim(spe)[1] 6623 46653REmove the spots that have not been manually assigned
sel= !is.na(colData(spe)$layer_guess_reordered)
spe = spe[,sel]
dim(spe)[1] 6623 46318normalization
spe <- computeLibraryFactors(spe)
spe <- logNormCounts(spe)Identification of the gene spatially relevvant
gene_i=NULL
pvalue_i=NULL
pvalue_mat=matrix(nrow=nrow(spe),ncol=length(sample_names))
for(i in 1:length(sample_names)){
sel=colData(spe)$sample_id==sample_names[i]
spe_sub= spe[,sel]
sparkX <- sparkx(logcounts(spe_sub),spatialCoords(spe_sub),numCores=1,option="mixture")
gene_i=c(gene_i,rowData(spe)$gene_id)
pvalue_i=c(pvalue_i,sparkX$res_mtest$combinedPval)
pvalue_mat[,i]=sparkX$res_mtest$combinedPval
print(sample_names[i])
}## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 4170
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5
[1] "151507"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 4285
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1!
Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151508"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 4708
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5
[1] "151509"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 4571
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1!
Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151510"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3587
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151669"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3274
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151670"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 4013
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5
[1] "151671"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3772
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151672"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3568
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5
[1] "151673"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3576
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5
[1] "151674"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3468
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151675"
## ===== SPARK-X INPUT INFORMATION ====
## number of total samples: 3326
## number of total genes: 6623
## Running with single core, may take some time
## Testing With Projection Kernel
## Testing With Gaussian Kernel 1
## Testing With Gaussian Kernel 2
## Testing With Gaussian Kernel 3
## Testing With Gaussian Kernel 4
## Testing With Gaussian Kernel 5
## Testing With Cosine Kernel 1
## Testing With Cosine Kernel 2
## Testing With Cosine Kernel 3
## Testing With Cosine Kernel 4
## Testing With Cosine Kernel 5Warning in FUN(newX[, i], ...): There are p-values that are exactly 1![1] "151676"oo=order(pvalue_i)
top_genes=gene_i[oo]
n=ave(1:length(top_genes), top_genes, FUN = seq_along)
top_genes=top_genes[n==1]
oo=order(apply(pvalue_mat,1,function(x) median(-log(x))),decreasing = TRUE)
top=gene_i[oo]PCA and HARMONY
data=t(logcounts(spe)[top[1:2000],])
subjects=colData(spe)$subject
dim(spe_sub)[1] 6623 3326 spe <- runPCA(spe, 50,subset_row = top[1:2000], scale=TRUE)
#pca=irlba(scale(data),50)$u
labels=as.factor(colData(spe)$layer_guess_reordered)
xy=as.matrix(spatialCoords(spe))
samples=colData(spe)$sample_id
spe <- RunHarmony(spe, "subject",lambda=NULL)
# pca <- RunHarmony(pca, subjects,lambda=NULL)
pca=reducedDim(spe,type = "HARMONY")[,1:50]
plot(pca)
KODAMA
kk=KODAMA.matrix.parallel(pca,
spatial = xy,
FUN= "PLS" ,
landmarks = 100000,
splitting = 100,
f.par.pls = 50,
spatial.resolution = 0.4,
n.cores=4)socket cluster with 4 nodes on host 'localhost'
================================================================================[1] "Finished parallel computation"
[1] "Calculation of dissimilarity matrix..."
================================================================================ print("KODAMA finished")[1] "KODAMA finished" config=umap.defaults
config$n_threads = 4
config$n_sgd_threads = "auto"
kk_UMAP=KODAMA.visualization(kk,method="UMAP",config=config)CLUSTER
library("mclust")
clu=kmeans(kk_UMAP,7,nstart = 100)$cluster
plot(kk_UMAP,col=labels,pch=20)
plot(kk_UMAP,col=clu,pch=20)
plot(xy,col=clu,pch=20)
u=unique(samples)
for(i in 1:length(u)){
sel=samples==u[i]
print(adjustedRandIndex(labels[sel],clu[sel]))
}[1] 0.5508229
[1] 0.4872925
[1] 0.5052779
[1] 0.494307
[1] 0.3409199
[1] 0.3167812
[1] 0.3902892
[1] 0.4299399
[1] 0.556732
[1] 0.5887864
[1] 0.5407068
[1] 0.5263028ref=refine_SVM(xy,clu,samples,cost=100)[1] "151507"
[1] "151508"
[1] "151509"
[1] "151510"
[1] "151669"
[1] "151670"
[1] "151671"
[1] "151672"
[1] "151673"
[1] "151674"
[1] "151675"
[1] "151676"u=unique(samples)
for(i in 1:length(u)){
sel=samples==u[i]
print(adjustedRandIndex(labels[sel],ref[sel]))
}[1] 0.5866949
[1] 0.5195753
[1] 0.5188101
[1] 0.5143312
[1] 0.3850269
[1] 0.3693473
[1] 0.4572459
[1] 0.5679025
[1] 0.6151074
[1] 0.6426947
[1] 0.6247849
[1] 0.6034484plot(xy,col=ref,pch=20)
TRAJECTORY
library("slingshot")
d <- slingshot(kk_UMAP, clusterLabels = clu)
trajectory=d@metadata$curves$Lineage1$s
k=knn_Armadillo(trajectory,kk_UMAP,1)
map_color=rainbow(nrow(trajectory))[k$nn_index]
plot(kk_UMAP,col=map_color)
sessionInfo()R version 4.4.0 (2024-04-24)
Platform: x86_64-pc-linux-gnu
Running under: Ubuntu 20.04.6 LTS
Matrix products: default
BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.9.0
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.9.0
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
time zone: Etc/UTC
tzcode source: system (glibc)
attached base packages:
[1] parallel stats4 stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] slingshot_2.12.0 TrajectoryUtils_1.12.0
[3] princurve_2.1.6 mclust_6.1.1
[5] KODAMAextra_1.0 e1071_1.7-14
[7] doParallel_1.0.17 iterators_1.0.14
[9] foreach_1.5.2 KODAMA_3.1
[11] umap_0.2.10.0 Rtsne_0.17
[13] minerva_1.5.10 spatialLIBD_1.16.2
[15] SpatialExperiment_1.14.0 Seurat_5.1.0
[17] SeuratObject_5.0.2 sp_2.1-4
[19] harmony_1.2.0 Rcpp_1.0.12
[21] SPARK_1.1.1 scry_1.16.0
[23] scran_1.32.0 scater_1.32.0
[25] ggplot2_3.5.1 scuttle_1.14.0
[27] SingleCellExperiment_1.26.0 SummarizedExperiment_1.34.0
[29] Biobase_2.64.0 GenomicRanges_1.56.1
[31] GenomeInfoDb_1.40.1 IRanges_2.38.1
[33] S4Vectors_0.42.1 BiocGenerics_0.50.0
[35] MatrixGenerics_1.16.0 matrixStats_1.3.0
[37] nnSVG_1.8.0 workflowr_1.7.1
loaded via a namespace (and not attached):
[1] goftest_1.2-3 DT_0.33
[3] Biostrings_2.72.1 vctrs_0.6.5
[5] spatstat.random_3.2-3 digest_0.6.36
[7] png_0.1-8 proxy_0.4-27
[9] git2r_0.33.0 ggrepel_0.9.5
[11] deldir_2.0-4 parallelly_1.37.1
[13] magick_2.8.3 MASS_7.3-61
[15] reshape2_1.4.4 httpuv_1.6.15
[17] withr_3.0.0 xfun_0.45
[19] survival_3.7-0 memoise_2.0.1
[21] benchmarkme_1.0.8 ggbeeswarm_0.7.2
[23] zoo_1.8-12 pbapply_1.7-2
[25] rematch2_2.1.2 KEGGREST_1.44.1
[27] promises_1.3.0 httr_1.4.7
[29] restfulr_0.0.15 globals_0.16.3
[31] fitdistrplus_1.1-11 ps_1.7.7
[33] rstudioapi_0.16.0 UCSC.utils_1.0.0
[35] miniUI_0.1.1.1 generics_0.1.3
[37] processx_3.8.4 curl_5.2.1
[39] fields_16.2 zlibbioc_1.50.0
[41] ScaledMatrix_1.12.0 polyclip_1.10-6
[43] doSNOW_1.0.20 GenomeInfoDbData_1.2.12
[45] ExperimentHub_2.12.0 SparseArray_1.4.8
[47] golem_0.4.1 xtable_1.8-4
[49] stringr_1.5.1 pracma_2.4.4
[51] evaluate_0.24.0 S4Arrays_1.4.1
[53] BiocFileCache_2.12.0 irlba_2.3.5.1
[55] colorspace_2.1-0 filelock_1.0.3
[57] ROCR_1.0-11 reticulate_1.38.0
[59] spatstat.data_3.1-2 shinyWidgets_0.8.6
[61] magrittr_2.0.3 lmtest_0.9-40
[63] later_1.3.2 viridis_0.6.5
[65] lattice_0.22-6 spatstat.geom_3.2-9
[67] future.apply_1.11.2 getPass_0.2-4
[69] scattermore_1.2 XML_3.99-0.17
[71] cowplot_1.1.3 RcppAnnoy_0.0.22
[73] class_7.3-22 pillar_1.9.0
[75] nlme_3.1-165 compiler_4.4.0
[77] beachmat_2.20.0 RSpectra_0.16-1
[79] stringi_1.8.4 tensor_1.5
[81] GenomicAlignments_1.40.0 plyr_1.8.9
[83] crayon_1.5.3 abind_1.4-5
[85] BiocIO_1.14.0 locfit_1.5-9.10
[87] bit_4.0.5 dplyr_1.1.4
[89] whisker_0.4.1 codetools_0.2-20
[91] BiocSingular_1.20.0 openssl_2.2.0
[93] bslib_0.7.0 paletteer_1.6.0
[95] plotly_4.10.4 mime_0.12
[97] splines_4.4.0 fastDummies_1.7.3
[99] dbplyr_2.5.0 sparseMatrixStats_1.16.0
[101] attempt_0.3.1 knitr_1.48
[103] blob_1.2.4 utf8_1.2.4
[105] BiocVersion_3.19.1 fs_1.6.4
[107] listenv_0.9.1 DelayedMatrixStats_1.26.0
[109] rdist_0.0.5 tibble_3.2.1
[111] Matrix_1.7-0 callr_3.7.6
[113] statmod_1.5.0 pkgconfig_2.0.3
[115] tools_4.4.0 BRISC_1.0.5
[117] cachem_1.1.0 RhpcBLASctl_0.23-42
[119] RSQLite_2.3.7 viridisLite_0.4.2
[121] DBI_1.2.3 fastmap_1.2.0
[123] rmarkdown_2.27 scales_1.3.0
[125] grid_4.4.0 ica_1.0-3
[127] Rsamtools_2.20.0 AnnotationHub_3.12.0
[129] sass_0.4.9 patchwork_1.2.0
[131] BiocManager_1.30.23 dotCall64_1.1-1
[133] RANN_2.6.1 snow_0.4-4
[135] yaml_2.3.9 rtracklayer_1.64.0
[137] cli_3.6.3 purrr_1.0.2
[139] leiden_0.4.3.1 lifecycle_1.0.4
[141] askpass_1.2.0 uwot_0.2.2
[143] bluster_1.14.0 sessioninfo_1.2.2
[145] BiocParallel_1.38.0 gtable_0.3.5
[147] rjson_0.2.21 ggridges_0.5.6
[149] progressr_0.14.0 limma_3.60.3
[151] jsonlite_1.8.8 edgeR_4.2.0
[153] RcppHNSW_0.6.0 bitops_1.0-7
[155] benchmarkmeData_1.0.4 bit64_4.0.5
[157] spatstat.utils_3.0-5 BiocNeighbors_1.22.0
[159] matlab_1.0.4.1 jquerylib_0.1.4
[161] highr_0.11 metapod_1.12.0
[163] config_0.3.2 dqrng_0.4.1
[165] lazyeval_0.2.2 shiny_1.8.1.1
[167] htmltools_0.5.8.1 sctransform_0.4.1
[169] rappdirs_0.3.3 glue_1.7.0
[171] spam_2.10-0 XVector_0.44.0
[173] RCurl_1.98-1.14 rprojroot_2.0.4
[175] gridExtra_2.3 igraph_2.0.3
[177] R6_2.5.1 tidyr_1.3.1
[179] CompQuadForm_1.4.3 cluster_2.1.6
[181] DelayedArray_0.30.1 tidyselect_1.2.1
[183] vipor_0.4.7 maps_3.4.2
[185] AnnotationDbi_1.66.0 future_1.33.2
[187] rsvd_1.0.5 munsell_0.5.1
[189] KernSmooth_2.23-24 data.table_1.15.4
[191] htmlwidgets_1.6.4 RColorBrewer_1.1-3
[193] rlang_1.1.4 spatstat.sparse_3.1-0
[195] spatstat.explore_3.2-7 fansi_1.0.6
[197] beeswarm_0.4.0