ChromVAR Failure to detect ATAC count data despite the data existing. #123
Description
Using the wrapper function RunChromVAR() fails to find the count data of a multiome Seurat object, RNA + ATAC, though when the object is checked, the data exists in the layer where RunChromVAR() expects to find it. I cannot tell if the issue is with ChromVAR or with RunChromVAR() but the function is failing to find the ATAC count data that I can see exists.
I am following the Seurat vignette found here: https://rdrr.io/github/satijalab/seurat/f/vignettes/weighted_nearest_neighbor_analysis.Rmd
Within the section "WNN analysis of 10x Multiome, RNA + ATAC",
The code works until RunChromVAR():
pbmc <- RunChromVAR( object = pbmc, genome = BSgenome.Hsapiens.UCSC.hg38 )
Which outputs the following error:
Computing GC bias per region Selecting background regions Computing deviations from background Constructing chromVAR assay **Warning: Layer counts isn't present in the assay object; returning NULL**
When I check the pbmc object:
pbmc
An object of class Seurat 167486 features across 10412 samples within 4 assays **Active assay: ATAC** (106056 features, 106056 variable features) **2 layers present: counts, data** 2 other assays present: RNA, SCT 5 dimensional reductions calculated: lsi, umap.atac, pca, umap.rna, wnn.umap
The ATAC layer is properly active and shows the presence of count data.
Looking at the structure of the ATAC count data shows it exists:
str(pbmc@assays$ATAC@counts)
Formal class 'dgCMatrix' [package "Matrix"] with 6 slots
..@ i : int [1:81240829] 12 16 25 27 28 30 34 37 38 39 ...
..@ p : int [1:10413] 0 13867 21114 27642 30965 35232 46860 54101 62700 70416 ...
..@ Dim : int [1:2] 106056 10412
..@ Dimnames:List of 2
.. ..$ : chr [1:106056] "chr1-10109-10357" "chr1-180730-181630" "chr1-191491-191736" "chr1-267816-268196" ...
.. ..$ : chr [1:10412] "AAACAGCCAAGGAATC-1" "AAACAGCCAATCCCTT-1" "AAACAGCCAATGCGCT-1" "AAACAGCCACCAACCG-1" ...
..@ x : num [1:81240829] 1 2 2 2 2 2 4 6 8 10 ...
..@ factors : list()
Double checking the existence of count data:
head(pbmc@assays$ATAC@counts)
6 x 10412 sparse Matrix of class "dgCMatrix"
[[ suppressing 10412 column names 'AAACAGCCAAGGAATC-1', 'AAACAGCCAATCCCTT-1', 'AAACAGCCAATGCGCT-1' ... ]]
chr1-10109-10357 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-180730-181630 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-191491-191736 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-267816-268196 . . . . . . . . . . . . . . . 2 . . . . . . . . . . 2 . . .
chr1-586028-586373 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
chr1-629721-630172 . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . .
I'm running this on the university HPC.
sessionInfo()
R version 4.4.0 (2024-04-24)
Platform: x86_64-pc-linux-gnu
Running under: CentOS Linux 7 (Core)
Matrix products: default
BLAS: /mmfs1/tools/R/gcc/9.3.0/4.4.0/lib64/R/lib/libRblas.so
LAPACK: /mmfs1/tools/R/gcc/9.3.0/4.4.0/lib64/R/lib/libRlapack.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=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: America/Chicago
tzcode source: system (glibc)
attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods
[8] base
other attached packages:
[1] BSgenome.Hsapiens.UCSC.hg38_1.4.5 BSgenome_1.72.0
[3] rtracklayer_1.64.0 BiocIO_1.14.0
[5] Biostrings_2.72.1 XVector_0.44.0
[7] motifmatchr_1.26.0 TFBSTools_1.42.0
[9] JASPAR2020_0.99.10 chromVAR_1.26.0
[11] biovizBase_1.52.0 ggplot2_3.5.1
[13] dplyr_1.1.4 EnsDb.Hsapiens.v86_2.99.0
[15] ensembldb_2.28.1 AnnotationFilter_1.28.0
[17] GenomicFeatures_1.56.0 AnnotationDbi_1.66.0
[19] Biobase_2.64.0 GenomicRanges_1.56.1
[21] GenomeInfoDb_1.40.1 IRanges_2.38.1
[23] S4Vectors_0.42.1 BiocGenerics_0.50.0
[25] Signac_1.14.0 Seurat_5.1.0
[27] SeuratObject_5.0.2 sp_2.1-4
loaded via a namespace (and not attached):
[1] ProtGenerics_1.36.0 matrixStats_1.5.0
[3] spatstat.sparse_3.1-0 bitops_1.0-9
[5] DirichletMultinomial_1.46.0 httr_1.4.7
[7] RColorBrewer_1.1-3 tools_4.4.0
[9] sctransform_0.4.1 backports_1.5.0
[11] R6_2.5.1 DT_0.33
[13] lazyeval_0.2.2 uwot_0.2.2
[15] withr_3.0.2 gridExtra_2.3
[17] progressr_0.15.1 cli_3.6.3
[19] spatstat.explore_3.3-4 fastDummies_1.7.5
[21] spatstat.data_3.1-4 readr_2.1.5
[23] ggridges_0.5.6 pbapply_1.7-2
[25] Rsamtools_2.20.0 foreign_0.8-86
[27] R.utils_2.12.3 dichromat_2.0-0.1
[29] parallelly_1.42.0 rstudioapi_0.17.1
[31] RSQLite_2.3.9 generics_0.1.3
[33] gtools_3.9.5 ica_1.0-3
[35] spatstat.random_3.3-2 GO.db_3.19.1
[37] Matrix_1.7-0 abind_1.4-8
[39] R.methodsS3_1.8.2 lifecycle_1.0.4
[41] yaml_2.3.10 SummarizedExperiment_1.34.0
[43] SparseArray_1.4.8 Rtsne_0.17
[45] glmGamPoi_1.16.0 grid_4.4.0
[47] blob_1.2.4 promises_1.3.2
[49] crayon_1.5.3 pwalign_1.0.0
[51] miniUI_0.1.1.1 lattice_0.22-6
[53] cowplot_1.1.3 annotate_1.82.0
[55] KEGGREST_1.44.1 pillar_1.10.1
[57] knitr_1.49 rjson_0.2.23
[59] future.apply_1.11.3 codetools_0.2-20
[61] fastmatch_1.1-6 leiden_0.4.3.1
[63] glue_1.8.0 spatstat.univar_3.1-1
[65] data.table_1.16.4 vctrs_0.6.5
[67] png_0.1-8 spam_2.11-1
[69] gtable_0.3.6 poweRlaw_0.80.0
[71] cachem_1.1.0 xfun_0.50
[73] S4Arrays_1.4.1 mime_0.12
[75] pracma_2.4.4 survival_3.5-8
[77] RcppRoll_0.3.1 fitdistrplus_1.2-2
[79] ROCR_1.0-11 nlme_3.1-164
[81] bit64_4.6.0-1 RcppAnnoy_0.0.22
[83] irlba_2.3.5.1 KernSmooth_2.23-22
[85] rpart_4.1.23 colorspace_2.1-1
[87] seqLogo_1.70.0 DBI_1.2.3
[89] Hmisc_5.2-3 nnet_7.3-19
[91] tidyselect_1.2.1 bit_4.5.0.1
[93] compiler_4.4.0 curl_6.2.0
[95] htmlTable_2.4.3 hdf5r_1.3.12
[97] DelayedArray_0.30.1 plotly_4.10.4
[99] checkmate_2.3.2 scales_1.3.0
[101] caTools_1.18.3 lmtest_0.9-40
[103] stringr_1.5.1 digest_0.6.37
[105] goftest_1.2-3 spatstat.utils_3.1-2
[107] rmarkdown_2.29 htmltools_0.5.8.1
[109] pkgconfig_2.0.3 base64enc_0.1-3
[111] sparseMatrixStats_1.16.0 MatrixGenerics_1.16.0
[113] fastmap_1.2.0 rlang_1.1.5
[115] htmlwidgets_1.6.4 UCSC.utils_1.0.0
[117] shiny_1.10.0 DelayedMatrixStats_1.26.0
[119] farver_2.1.2 zoo_1.8-12
[121] jsonlite_1.8.9 BiocParallel_1.38.0
[123] R.oo_1.27.0 VariantAnnotation_1.50.0
[125] RCurl_1.98-1.16 magrittr_2.0.3
[127] Formula_1.2-5 GenomeInfoDbData_1.2.12
[129] dotCall64_1.2 patchwork_1.3.0
[131] munsell_0.5.1 Rcpp_1.0.14
[133] reticulate_1.40.0 stringi_1.8.4
[135] zlibbioc_1.50.0 MASS_7.3-60.2
[137] plyr_1.8.9 parallel_4.4.0
[139] listenv_0.9.1 ggrepel_0.9.6
[141] deldir_2.0-4 CNEr_1.40.0
[143] splines_4.4.0 tensor_1.5
[145] hms_1.1.3 igraph_2.1.4
[147] spatstat.geom_3.3-5 RcppHNSW_0.6.0
[149] reshape2_1.4.4 TFMPvalue_0.0.9
[151] XML_3.99-0.18 evaluate_1.0.3
[153] tzdb_0.5.0 httpuv_1.6.15
[155] RANN_2.6.2 tidyr_1.3.1
[157] purrr_1.0.2 polyclip_1.10-7
[159] future_1.34.0 scattermore_1.2
[161] xtable_1.8-4 restfulr_0.0.15
[163] RSpectra_0.16-2 later_1.4.1
[165] viridisLite_0.4.2 tibble_3.2.1
[167] memoise_2.0.1 GenomicAlignments_1.40.0
[169] cluster_2.1.6 globals_0.16.3
Vignette of reproducible code is found here: https://rdrr.io/github/satijalab/seurat/f/vignettes/weighted_nearest_neighbor_analysis.Rmd