Introduction to spatialDE
Introduction
SpatialDE by Svensson et al., 2018, is a method to identify spatially variable genes (SVGs) in spatially resolved transcriptomics data.
Installation
You can install spatialDE from Bioconductor with the following code:
Example: Mouse Olfactory Bulb
Reproducing the example analysis from the original SpatialDE Python package.
Load data
Files originally retrieved from SpatialDE GitHub repository from the following links: https://github.com/Teichlab/SpatialDE/blob/master/Analysis/MouseOB/data/Rep11_MOB_0.csv https://github.com/Teichlab/SpatialDE/blob/master/Analysis/MouseOB/MOB_sample_info.csv
# Expression file used in python SpatialDE.
data("Rep11_MOB_0")
# Sample Info file used in python SpatialDE
data("MOB_sample_info")The Rep11_MOB_0 object contains spatial expression data
for 16218 genes on 262 spots, with genes as rows and spots as
columns.
Rep11_MOB_0[1:5, 1:5]
#> 16.92x9.015 16.945x11.075 16.97x10.118 16.939x12.132 16.949x13.055
#> Nrf1 1 0 0 1 0
#> Zbtb5 1 0 1 0 0
#> Ccnl1 1 3 1 1 0
#> Lrrfip1 2 2 0 0 3
#> Bbs1 1 2 0 4 0
dim(Rep11_MOB_0)
#> [1] 16218 262The MOB_sample_info object contains a
data.frame with coordinates for each spot.
head(MOB_sample_info)
#> x y total_counts
#> 16.92x9.015 16.920 9.015 18790
#> 16.945x11.075 16.945 11.075 36990
#> 16.97x10.118 16.970 10.118 12471
#> 16.939x12.132 16.939 12.132 22703
#> 16.949x13.055 16.949 13.055 18641
#> 16.942x15.088 16.942 15.088 23408Get total_counts for every spot
Rep11_MOB_0 <- Rep11_MOB_0[, row.names(MOB_sample_info)]
MOB_sample_info$total_counts <- colSums(Rep11_MOB_0)
head(MOB_sample_info)
#> x y total_counts
#> 16.92x9.015 16.920 9.015 18790
#> 16.945x11.075 16.945 11.075 36990
#> 16.97x10.118 16.970 10.118 12471
#> 16.939x12.132 16.939 12.132 22703
#> 16.949x13.055 16.949 13.055 18641
#> 16.942x15.088 16.942 15.088 23408stabilize
The SpatialDE method assumes normally distributed data, so we
stabilize the variance of the negative binomial distributed counts data
using Anscombe’s approximation. The stabilize() function
takes as input a data.frame of expression values with
samples in columns and genes in rows. Thus, in this case, we have to
transpose the data.
norm_expr <- stabilize(Rep11_MOB_0)
#> + /github/home/.cache/R/basilisk/1.17.2/0/bin/conda create --yes --prefix /github/home/.cache/R/basilisk/1.17.2/spatialDE/1.11.0/env 'python=3.10.14' --quiet -c conda-forge --override-channels
#> + /github/home/.cache/R/basilisk/1.17.2/0/bin/conda install --yes --prefix /github/home/.cache/R/basilisk/1.17.2/spatialDE/1.11.0/env 'python=3.10.14' -c conda-forge --override-channels
#> + /github/home/.cache/R/basilisk/1.17.2/0/bin/conda install --yes --prefix /github/home/.cache/R/basilisk/1.17.2/spatialDE/1.11.0/env -c conda-forge 'python=3.10.14' 'numpy=1.23.5' 'scipy=1.9.3' 'patsy=0.5.3' 'pandas=1.5.2' --override-channels
norm_expr[1:5, 1:5]
#> 16.92x9.015 16.945x11.075 16.97x10.118 16.939x12.132 16.949x13.055
#> Nrf1 1.227749 0.8810934 0.8810934 1.2277491 0.8810934
#> Zbtb5 1.227749 0.8810934 1.2277491 0.8810934 0.8810934
#> Ccnl1 1.227749 1.6889027 1.2277491 1.2277491 0.8810934
#> Lrrfip1 1.484676 1.4846765 0.8810934 0.8810934 1.6889027
#> Bbs1 1.227749 1.4846765 0.8810934 1.8584110 0.8810934regress_out
Next, we account for differences in library size between the samples by regressing out the effect of the total counts for each gene using linear regression.
resid_expr <- regress_out(norm_expr, sample_info = MOB_sample_info)
resid_expr[1:5, 1:5]
#> 16.92x9.015 16.945x11.075 16.97x10.118 16.939x12.132 16.949x13.055
#> Nrf1 -0.75226761 -1.2352000 -1.0164479 -0.7903289 -1.0973214
#> Zbtb5 0.09242373 -0.3323719 0.1397144 -0.2760560 -0.2533134
#> Ccnl1 -2.77597164 -2.5903783 -2.6092013 -2.8529340 -3.1193883
#> Lrrfip1 -1.92331333 -2.1578718 -2.3849405 -2.5924072 -1.7163300
#> Bbs1 -1.12186064 -1.0266476 -1.3706460 -0.5363646 -1.4666155run
To reduce running time, the SpatialDE test is run on a subset of 1000 genes. Running it on the complete data set takes about 10 minutes.
# For this example, run spatialDE on the first 1000 genes
sample_resid_expr <- head(resid_expr, 1000)
results <- spatialDE::run(sample_resid_expr, coordinates = X)
head(results[order(results$qval), ])
#> FSV M g l max_delta max_ll max_mu_hat max_s2_t_hat
#> 752 0.5869020 4 Fabp7 1.135190 0.6872704 -154.7691 5.036316 3.6039400
#> 671 0.5276876 4 Apc 1.135190 0.8739619 -146.9456 -3.922771 2.1552117
#> 884 0.3651030 4 Kcnh3 1.907609 1.6225593 -177.5658 -8.066532 3.7081686
#> 668 0.3837007 4 Pcp4 1.135190 1.5683362 -127.1354 -11.866414 16.9252387
#> 722 0.3345161 4 Penk 1.135190 1.9424986 -163.0520 -9.491798 10.3640716
#> 725 0.3931224 4 Frzb 1.135190 1.5073475 -217.1284 1.180284 0.3109513
#> model n s2_FSV s2_logdelta time BIC max_ll_null
#> 752 SE 260 0.0004068815 0.007999172 0.001466274 331.7809 -196.7353
#> 671 SE 260 0.0004218510 0.007939872 0.001480341 316.1339 -178.4892
#> 884 SE 260 0.0006305732 0.013314547 0.001743317 377.3743 -200.6982
#> 668 SE 260 0.0007326393 0.014985240 0.001722813 276.5136 -144.3654
#> 722 SE 260 0.0006993170 0.015755817 0.001959801 348.3468 -179.1260
#> 725 SE 260 0.0009077964 0.018308366 0.001463652 456.4996 -233.0538
#> LLR pval qval
#> 752 41.96623 9.286349e-11 9.286349e-08
#> 671 31.54359 1.950129e-08 9.750644e-06
#> 884 23.13242 1.512193e-06 5.040644e-04
#> 668 17.22998 3.311683e-05 8.279207e-03
#> 722 16.07398 6.091542e-05 1.098165e-02
#> 725 15.92536 6.588988e-05 1.098165e-02model_search
Finally, we can classify the DE genes to interpetable DE classes
using the model_search function. We apply the model search
on filtered DE results, using a threshold of 0.05 for the Q-values.
de_results <- results[results$qval < 0.05, ]
ms_results <- model_search(
sample_resid_expr,
coordinates = X,
de_results = de_results
)
# To show ms_results sorted on qvalue, uncomment the following line
# head(ms_results[order(ms_results$qval), ])
head(ms_results)
#> BIC FSV LLR M g l max_delta max_ll
#> 0 273.4206 0.06585344 NaN 4 X2900097C17Rik 5.386796 14.481719 -125.5890
#> 1 267.1570 0.10520888 NaN 4 Pcp4 9.052138 8.896112 -122.4571
#> 2 333.8619 0.10540242 NaN 4 Rbfox3 9.052138 8.877856 -155.8096
#> 3 370.2613 0.09234754 NaN 4 Sez6 9.052138 10.280760 -174.0093
#> 4 378.8001 0.08317351 NaN 4 Gng13 9.052138 11.530100 -178.2787
#> 5 394.8754 0.10227380 NaN 4 Gng4 9.052138 9.181433 -186.3163
#> max_mu_hat max_s2_t_hat model n s2_FSV s2_logdelta time
#> 0 -5.162205 1.808970 PER 260 0.0001697748 0.03696936 0.004333496
#> 1 -11.838117 14.729972 PER 260 0.0004604459 0.04570537 0.001976490
#> 2 -9.007761 8.556802 PER 260 0.0004796162 0.04746842 0.001969814
#> 3 -10.734952 10.511043 PER 260 0.0005243492 0.06432634 0.001965523
#> 4 3.970898 1.300596 PER 260 0.0004296904 0.06273417 0.001996517
#> 5 -12.350677 15.549625 PER 260 0.0005103723 0.05301686 0.001957417
#> PER_prob SE_prob linear_prob pval qval max_ll_null
#> 0 0.8657858 1.342142e-01 3.671512e-10 3.776266e-04 0.026973327 -139.1608
#> 1 0.9999136 8.638788e-05 2.434950e-17 3.311683e-05 0.008279207 -144.3654
#> 2 1.0000000 4.526865e-08 1.223837e-18 1.105005e-04 0.013812568 -179.2132
#> 3 0.9997843 2.156997e-04 2.311672e-12 5.444193e-04 0.036294619 -190.1870
#> 4 0.7364092 2.635908e-01 3.356690e-12 9.052709e-05 0.012932442 -194.1171
#> 5 0.9999908 9.156503e-06 9.984359e-15 2.953494e-04 0.023714017 -205.2165spatial_patterns
Furthermore, we can group spatially variable genes (SVGs) into spatial patterns using automatic expression histology (AEH).
sp <- spatial_patterns(
sample_resid_expr,
coordinates = X,
de_results = de_results,
n_patterns = 4L, length = 1.5
)
sp$pattern_results
#> g pattern membership
#> 595 X2900097C17Rik 3 1
#> 660 Eef1a1 0 1
#> 667 Pmepa1 2 1
#> 668 Pcp4 0 1
#> 671 Apc 2 1
#> 696 Rbfox3 0 1
#> 722 Penk 0 1
#> 725 Frzb 2 1
#> 752 Fabp7 2 1
#> 791 Gm13889 0 1
#> 808 Sez6 0 1
#> 829 Gng13 2 1
#> 833 Gng4 0 1
#> 884 Kcnh3 0 1
#> 906 Camk2b 0 1Plots
Visualizing one of the most significant genes.
gene <- "Pcp4"
ggplot(data = MOB_sample_info, aes(x = x, y = y, color = norm_expr[gene, ])) +
geom_point(size = 7) +
ggtitle(gene) +
scale_color_viridis_c() +
labs(color = gene)
Plot Spatial Patterns of Multiple Genes
As an alternative to the previous figure, we can plot multiple genes using the normalized expression values.
ordered_de_results <- de_results[order(de_results$qval), ]
multiGenePlots(norm_expr,
coordinates = X,
ordered_de_results[1:6, ]$g,
point_size = 4,
viridis_option = "D",
dark_theme = FALSE
)
SpatialExperiment integration
The SpatialDE workflow can also be executed with a SpatialExperiment object as input.
library(SpatialExperiment)
# For SpatialExperiment object, we neeed to transpose the counts matrix in order
# to have genes on rows and spot on columns.
# For this example, run spatialDE on the first 1000 genes
partial_counts <- head(Rep11_MOB_0, 1000)
spe <- SpatialExperiment(
assays = list(counts = partial_counts),
spatialData = DataFrame(MOB_sample_info[, c("x", "y")]),
spatialCoordsNames = c("x", "y")
)
out <- spatialDE(spe, assay_type = "counts", verbose = FALSE)
head(out[order(out$qval), ])
#> FSV M g l max_delta max_ll max_mu_hat max_s2_t_hat
#> 759 0.5853287 4 Fabp7 1.135190 0.6917423 -157.8641 4.805770 3.2961235
#> 684 0.5215592 4 Apc 1.135190 0.8957046 -152.4253 -2.852122 1.1971964
#> 886 0.3425473 4 Kcnh3 1.907609 1.7908397 -201.1959 -7.162902 2.9230555
#> 681 0.3671680 4 Pcp4 1.135190 1.6829213 -124.8416 -9.209852 10.0688118
#> 735 0.3820350 4 Frzb 1.135190 1.5794319 -239.8355 1.156500 0.3240445
#> 832 0.2378540 4 Gng13 1.907609 2.9897888 -204.1019 3.648425 0.7506023
#> model n s2_FSV s2_logdelta time BIC max_ll_null
#> 759 SE 260 0.0004113112 0.008071972 0.001455069 337.9709 -199.4754
#> 684 SE 260 0.0004271087 0.008023534 0.001578331 327.0933 -183.5046
#> 886 SE 260 0.0006874927 0.015203449 0.001739264 424.6346 -221.4201
#> 681 SE 260 0.0008102845 0.017044171 0.001735449 271.9259 -140.0619
#> 735 SE 260 0.0009344367 0.019163063 0.001512289 501.9136 -255.0426
#> 832 SE 260 0.0008862383 0.027973295 0.002026320 430.4466 -218.4267
#> LLR pval qval
#> 759 41.61134 1.113458e-10 1.113458e-07
#> 684 31.07932 2.476965e-08 1.238482e-05
#> 886 20.22416 6.887758e-06 2.295919e-03
#> 681 15.22028 9.567033e-05 1.926777e-02
#> 735 15.20713 9.633886e-05 1.926777e-02
#> 832 14.32476 1.538286e-04 2.563810e-02Plot Spatial Patterns of Multiple Genes (using SpatialExperiment object)
We can plot spatial patterns of multiples genes using the
spe object.
spe_results <- out[out$qval < 0.05, ]
ordered_spe_results <- spe_results[order(spe_results$qval), ]
multiGenePlots(spe,
assay_type = "counts",
ordered_spe_results[1:6, ]$g,
point_size = 4,
viridis_option = "D",
dark_theme = FALSE
)
Classify spatially variable genes with model_search and
spatial_patterns
msearch <- modelSearch(spe,
de_results = out, qval_thresh = 0.05,
verbose = FALSE
)
head(msearch)
#> BIC FSV LLR M g l max_delta max_ll
#> 0 287.6679 0.06406448 NaN 4 X2900097C17Rik 5.386796 14.914618 -132.7126
#> 1 263.5043 0.09590705 NaN 4 Pcp4 9.052138 9.860377 -120.6308
#> 2 370.8671 0.09681127 NaN 4 Rbfox3 9.052138 9.758510 -174.3122
#> 3 428.9359 0.08063297 NaN 4 Gng13 9.052138 11.926341 -203.3466
#> 4 401.7737 0.14867725 NaN 4 Kcnh3 9.052138 5.989364 -189.7655
#> 5 479.4804 0.36943862 14.01272 4 Pmepa1 1.135190 1.666576 -228.6188
#> max_mu_hat max_s2_t_hat model n s2_FSV s2_logdelta time
#> 0 -3.6572048 0.8870009 PER 260 0.0001665651 0.03805707 0.007774353
#> 1 -9.1839788 8.0150340 PER 260 0.0004344069 0.05008671 0.002029181
#> 2 -7.7847610 5.8291532 PER 260 0.0004545203 0.05160913 0.002008677
#> 3 3.6145343 1.0492566 PER 260 0.0004240747 0.06523479 0.001991510
#> 4 -7.1054452 7.8745493 PER 260 0.0006681386 0.03907065 0.002011776
#> 5 0.1238686 0.1463362 SE 260 0.0007025967 0.01471845 0.001704216
#> PER_prob SE_prob linear_prob pval qval max_ll_null
#> 0 0.55766485 4.423351e-01 5.814904e-10 3.104656e-04 0.032188921 -145.8346
#> 1 0.99977999 2.200120e-04 3.452128e-15 9.567033e-05 0.019267772 -140.0619
#> 2 0.99999990 1.031935e-07 1.553124e-16 3.218892e-04 0.032188921 -195.2940
#> 3 0.81916872 1.808313e-01 1.701216e-11 1.538286e-04 0.025638100 -218.4267
#> 4 1.00000000 1.179426e-10 8.336815e-26 6.887758e-06 0.002295919 -221.4201
#> 5 0.03133507 9.686649e-01 1.700936e-10 1.815779e-04 0.025717859 -242.6315spatterns <- spatialPatterns(spe,
de_results = de_results, qval_thresh = 0.05,
n_patterns = 4L, length = 1.5, verbose = FALSE
)
spatterns$pattern_results
#> g pattern membership
#> 595 X2900097C17Rik 1 1
#> 660 Eef1a1 1 1
#> 667 Pmepa1 3 1
#> 668 Pcp4 0 1
#> 671 Apc 3 1
#> 696 Rbfox3 0 1
#> 722 Penk 0 1
#> 725 Frzb 3 1
#> 752 Fabp7 3 1
#> 791 Gm13889 0 1
#> 808 Sez6 0 1
#> 829 Gng13 3 1
#> 833 Gng4 2 1
#> 884 Kcnh3 0 1
#> 906 Camk2b 0 1sessionInfo
Session info
#> [1] "2024-08-29 03:23:14 UTC"
#> R version 4.4.1 (2024-06-14)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04 LTS
#>
#> Matrix products: default
#> BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
#> LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=C
#> [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] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] SpatialExperiment_1.15.1 SingleCellExperiment_1.27.2
#> [3] SummarizedExperiment_1.35.1 Biobase_2.65.1
#> [5] GenomicRanges_1.57.1 GenomeInfoDb_1.41.1
#> [7] IRanges_2.39.2 S4Vectors_0.43.2
#> [9] BiocGenerics_0.51.0 MatrixGenerics_1.17.0
#> [11] matrixStats_1.3.0 ggplot2_3.5.1
#> [13] spatialDE_1.11.0 BiocStyle_2.33.1
#>
#> loaded via a namespace (and not attached):
#> [1] gtable_0.3.5 dir.expiry_1.13.0 rjson_0.2.22
#> [4] xfun_0.47 bslib_0.8.0 ggrepel_0.9.5
#> [7] lattice_0.22-6 vctrs_0.6.5 tools_4.4.1
#> [10] generics_0.1.3 parallel_4.4.1 tibble_3.2.1
#> [13] fansi_1.0.6 highr_0.11 pkgconfig_2.0.3
#> [16] Matrix_1.7-0 checkmate_2.3.2 lifecycle_1.0.4
#> [19] GenomeInfoDbData_1.2.12 farver_2.1.2 compiler_4.4.1
#> [22] munsell_0.5.1 htmltools_0.5.8.1 sys_3.4.2
#> [25] buildtools_1.0.0 sass_0.4.9 yaml_2.3.10
#> [28] pillar_1.9.0 crayon_1.5.3 jquerylib_0.1.4
#> [31] cachem_1.1.0 DelayedArray_0.31.11 magick_2.8.4
#> [34] abind_1.4-5 basilisk_1.17.2 tidyselect_1.2.1
#> [37] digest_0.6.37 dplyr_1.1.4 labeling_0.4.3
#> [40] maketools_1.3.0 fastmap_1.2.0 grid_4.4.1
#> [43] colorspace_2.1-1 cli_3.6.3 SparseArray_1.5.31
#> [46] magrittr_2.0.3 S4Arrays_1.5.7 utf8_1.2.4
#> [49] withr_3.0.1 backports_1.5.0 filelock_1.0.3
#> [52] scales_1.3.0 UCSC.utils_1.1.0 rmarkdown_2.28
#> [55] XVector_0.45.0 httr_1.4.7 gridExtra_2.3
#> [58] reticulate_1.38.0 png_0.1-8 evaluate_0.24.0
#> [61] knitr_1.48 basilisk.utils_1.17.2 viridisLite_0.4.2
#> [64] rlang_1.1.4 Rcpp_1.0.13 glue_1.7.0
#> [67] BiocManager_1.30.25 jsonlite_1.8.8 R6_2.5.1
#> [70] zlibbioc_1.51.1