SpatialPlot not working as its supposed to

[Aditya-Gautam20]

Previously ,

SpatialPlot(sample_1, group.by = "cluster_annotations") ,

used to give image

now i am getting no spots , please help

[samuel-marsh]

Hi,

Not member of dev team but hopefully can be helpful.
Couple things to try.

1. Run UpdateSeuratObject just to check all the object structure is ok.
   If issue persists then:
2. I would recommend trying uninstalling and reinstalling Seurat, SeuratObject, and ggplot2 (just to rule out issue in installation).

If the issue still persists can you provide reproducible example using dataset from SeuratData package (to rule out object specific issue) and the output of sessionInfo()?

Best,
Sam

[Aditya-Gautam20]

Hello Sam ,

Thankyou for helping ,I tried 1,2 issue still there , below is the session info

Regards
Aditya Gautam

sessionInfo()
R version 4.3.1 (2023-06-16)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Pop!_OS 22.04 LTS

Matrix products: default
BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.10.0
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0

locale:
[1] LC_CTYPE=en_IN.UTF-8 LC_NUMERIC=C LC_TIME=en_IN.UTF-8
[4] LC_COLLATE=en_IN.UTF-8 LC_MONETARY=en_IN.UTF-8 LC_MESSAGES=en_IN.UTF-8
[7] LC_PAPER=en_IN.UTF-8 LC_NAME=C LC_ADDRESS=C
[10] LC_TELEPHONE=C LC_MEASUREMENT=en_IN.UTF-8 LC_IDENTIFICATION=C

time zone: Asia/Kolkata
tzcode source: system (glibc)

attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods base

other attached packages:
[1] GSEABase_1.62.0 graph_1.78.0 annotate_1.78.0
[4] XML_3.99-0.17 AnnotationDbi_1.64.1 singscore_1.19.1
[7] EnhancedVolcano_1.18.0 ggrepel_0.9.6 affy_1.78.2
[10] immunedeconv_2.1.0 EPIC_1.1.7 estimate_1.0.13
[13] msigdbr_7.5.1 limma_3.56.2 data.table_1.16.2
[16] fgsea_1.26.0 Seurat_5.1.0 SeuratObject_5.0.2
[19] sp_2.1-4 RColorBrewer_1.1-3 pheatmap_1.0.12
[22] vsn_3.68.0 lubridate_1.9.3 forcats_1.0.0
[25] stringr_1.5.1 purrr_1.0.2 tidyr_1.3.1
[28] tibble_3.2.1 tidyverse_2.0.0 common_1.1.3
[31] gridExtra_2.3 readr_2.1.5 DT_0.33
[34] dplyr_1.1.4 DESeq2_1.40.2 SummarizedExperiment_1.32.0
[37] Biobase_2.62.0 MatrixGenerics_1.14.0 matrixStats_1.4.1
[40] GenomicRanges_1.54.1 GenomeInfoDb_1.38.2 IRanges_2.36.0
[43] S4Vectors_0.40.2 BiocGenerics_0.48.1 survminer_0.5.0
[46] ggpubr_0.6.0 ggplot2_3.5.1 survival_3.7-0
[49] gplots_3.2.0 CIBERSORT_0.1.0 GenomicDataCommons_1.24.3
[52] magrittr_2.0.3 TCGAbiolinks_2.28.4

loaded via a namespace (and not attached):
[1] spatstat.sparse_3.1-0 bitops_1.0-9 httr_1.4.7
[4] tools_4.3.1 sctransform_0.4.1 backports_1.5.0
[7] utf8_1.2.4 R6_2.5.1 mgcv_1.9-0
[10] mMCPcounter_1.1.0 uwot_0.2.2 lazyeval_0.2.2
[13] withr_3.0.2 prettyunits_1.2.0 preprocessCore_1.62.1
[16] progressr_0.15.0 cli_3.6.3 spatstat.explore_3.3-3
[19] fastDummies_1.7.4 labeling_0.4.3 ComICS_1.0.4
[22] survMisc_0.5.6 spatstat.data_3.1-2 genefilter_1.82.1
[25] proxy_0.4-27 ggridges_0.5.6 pbapply_1.7-2
[28] parallelly_1.38.0 readxl_1.4.3 rstudioapi_0.17.1
[31] RSQLite_2.3.7 generics_0.1.3 testit_0.13
[34] gtools_3.9.5 ica_1.0-3 spatstat.random_3.3-2
[37] car_3.1-3 Matrix_1.6-5 fansi_1.0.6
[40] abind_1.4-8 lifecycle_1.0.4 edgeR_3.42.4
[43] carData_3.0-5 SparseArray_1.2.2 BiocFileCache_2.10.1
[46] Rtsne_0.17 grid_4.3.1 blob_1.2.4
[49] promises_1.3.0 crayon_1.5.3 miniUI_0.1.1.1
[52] lattice_0.22-5 cowplot_1.1.3 KEGGREST_1.42.0
[55] pillar_1.9.0 knitr_1.48 TCGAbiolinksGUI.data_1.20.0
[58] future.apply_1.11.3 codetools_0.2-19 fastmatch_1.1-4
[61] leiden_0.4.3.1 glue_1.8.0 downloader_0.4
[64] spatstat.univar_3.1-1 vctrs_0.6.5 png_0.1-8
[67] spam_2.11-0 cellranger_1.1.0 gtable_0.3.6
[70] cachem_1.1.0 xfun_0.49 S4Arrays_1.2.0
[73] mime_0.12 KMsurv_0.1-5 fitdistrplus_1.2-1
[76] ROCR_1.0-11 nlme_3.1-163 bit64_4.5.2
[79] progress_1.2.3 filelock_1.0.3 RcppAnnoy_0.0.22
[82] data.tree_1.1.0 affyio_1.72.0 irlba_2.3.5.1
[85] KernSmooth_2.23-22 colorspace_2.1-1 DBI_1.2.3
[88] tidyselect_1.2.1 bit_4.5.0 compiler_4.3.1
[91] curl_6.0.0 rvest_1.0.4 xml2_1.3.6
[94] DelayedArray_0.28.0 plotly_4.10.4 scales_1.3.0
[97] caTools_1.18.3 lmtest_0.9-40 rappdirs_0.3.3
[100] goftest_1.2-3 digest_0.6.37 spatstat.utils_3.1-1
[103] XVector_0.42.0 htmltools_0.5.8.1 pkgconfig_2.0.3
[106] dbplyr_2.5.0 fastmap_1.2.0 rlang_1.1.4
[109] htmlwidgets_1.6.4 shiny_1.9.1 farver_2.1.2
[112] zoo_1.8-12 jsonlite_1.8.9 BiocParallel_1.36.0
[115] RCurl_1.98-1.16 Formula_1.2-5 GenomeInfoDbData_1.2.11
[118] dotCall64_1.2 patchwork_1.3.0 munsell_0.5.1
[121] Rcpp_1.0.13-1 babelgene_22.9 reticulate_1.39.0
[124] furrr_0.3.1 stringi_1.8.4 zlibbioc_1.48.0
[127] MASS_7.3-60 plyr_1.8.9 parallel_4.3.1
[130] listenv_0.9.1 deldir_2.0-4 Biostrings_2.70.1
[133] splines_4.3.1 tensor_1.5 hms_1.1.3
[136] locfit_1.5-9.10 igraph_2.1.1 spatstat.geom_3.3-3
[139] ggsignif_0.6.4 RcppHNSW_0.6.0 reshape2_1.4.4
[142] biomaRt_2.58.0 BiocManager_1.30.25 tzdb_0.4.0
[145] httpuv_1.6.15 RANN_2.6.2 polyclip_1.10-7
[148] future_1.34.0 km.ci_0.5-6 scattermore_1.2
[151] broom_1.0.7 xtable_1.8-4 e1071_1.7-16
[154] RSpectra_0.16-2 rstatix_0.7.2 later_1.3.2
[157] viridisLite_0.4.2 class_7.3-22 memoise_2.0.1
[160] cluster_2.1.6 sva_3.48.0 timechange_0.3.0
[163] globals_0.16.3

[samuel-marsh]

Are you able to provide reproducible example from SeuratData? I’m unable to reproduce your issue.

Best,
Sam

[Aditya-Gautam20]

Hello Sam

Thanks for the help, below is the file used and the function i'm running, will you be able to reproduce the error now?

Regards
Aditya Gautam

sample_1_zip.zip

load("path to sample1")
SpatialPlot(sample_1, group.by = "cluster_annotations")

[samuel-marsh]

Hi,

Can you please try and replicate issue with object that is known to be good. Here is example using the code from SeuratData and basic spatial vignette.

Best,
Sam

suppressMessages(library(Seurat))
suppressMessages(library(SeuratData))
suppressMessages(library(ggplot2))
suppressMessages(library(patchwork))
#> Warning: package 'patchwork' was built under R version 4.4.1
suppressMessages(library(dplyr))

brain <- LoadData("stxBrain", type = "anterior1")
#> Validating object structure
#> Updating object slots
#> Ensuring keys are in the proper structure
#> Ensuring keys are in the proper structure
#> Ensuring feature names don't have underscores or pipes
#> Updating slots in Spatial
#> Updating slots in anterior1
#> Validating object structure for Assay5 'Spatial'
#> Validating object structure for VisiumV2 'anterior1'
#> Object representation is consistent with the most current Seurat version
brain <- SCTransform(brain, assay = "Spatial", verbose = FALSE)

brain <- RunPCA(brain, assay = "SCT", verbose = FALSE)
brain <- FindNeighbors(brain, reduction = "pca", dims = 1:30)
#> Computing nearest neighbor graph
#> Computing SNN
brain <- FindClusters(brain, verbose = FALSE)
brain <- suppressMessages(RunUMAP(brain, reduction = "pca", dims = 1:30))
#> Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
#> To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
#> This message will be shown once per session

p1 <- DimPlot(brain, reduction = "umap", label = TRUE)
p2 <- SpatialDimPlot(brain, label = TRUE, label.size = 3, pt.size.factor = 2)
#> Scale for fill is already present.
#> Adding another scale for fill, which will replace the existing scale.
p1 + p2

^{Created on 2024-11-08 with reprex v2.1.1}

Session info

sessioninfo::session_info()
#> ─ Session info ───────────────────────────────────────────────────────────────
#>  setting  value
#>  version  R version 4.4.0 (2024-04-24)
#>  os       macOS Monterey 12.7.5
#>  system   x86_64, darwin20
#>  ui       X11
#>  language (EN)
#>  collate  en_US.UTF-8
#>  ctype    en_US.UTF-8
#>  tz       America/New_York
#>  date     2024-11-08
#>  pandoc   3.2 @ /Applications/RStudio.app/Contents/Resources/app/quarto/bin/tools/x86_64/ (via rmarkdown)
#> 
#> ─ Packages ───────────────────────────────────────────────────────────────────
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#>  BiocGenerics                0.50.0     2024-04-30 [1] Bioconductor 3.19 (R 4.4.0)
#>  bmcite.SeuratData         * 0.3.0      2024-05-01 [1] local
#>  cli                         3.6.3      2024-06-21 [1] CRAN (R 4.4.0)
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[Aditya-Gautam20]

I ran this same code , i was able to replicate the results here , but for my data its not working

[samuel-marsh]

Ok great. Well that narrows things down to an issue with your object and not Seurat generally. I would next suggest re-creating the object from the raw data to see if that solves the issue.

Best,
Sam

[longmanz]

Hi @Aditya-Gautam20 ,
As Sam suggested, your Seurat object seems not correctly generated. It only contains a SCT assay and I cannot find the raw RNA assay.

Could you try to re-generate your object again and see if it works? Or you can share the commands that you used to generate your object with me so I can look into this.