PercentageFeatureSet error

[iva302]

Hello,
I am running the following code and it fails to add percent.mt information to my seurat object. I have performed this analysis successfully on the same sample before. I have checked the structure of all the objects and everything looks good. This is mouse sample.

the 10x hdf5 file contains both data types.

inputdata.10x <- Read10X_h5("/mnt/Disk2/iva/e8.5_multiome/1180A/outs/filtered_feature_bc_matrix.h5")

extract RNA and ATAC data

rna_counts <- inputdata.10x$Gene Expression
wt <- CreateSeuratObject(counts = rna_counts)
wt[["percent.mt"]] <- PercentageFeatureSet(wt, pattern = "^mt-")

This is the error message-
......."Gm28406", "Gm29436", "Gm28977", "Gm28407", "Gm29392", "Gm29393", "Gm21294",
"Gm28672", "Gm28670", "Gm28673", "Gm28674", "Gm21996", "Gm28930", "Gm29504",
"Gm20837", "Gm28300", "Gm28301", "Gm21860", "Gm47283", "Gm21748", "mt-Nd1",
"mt-Nd2", "mt-Co1", "mt-Co2", "mt-Atp8", "mt-Atp6", "mt-Co3", "mt-Nd3", "mt-Nd4l",
"mt-Nd4", "mt-Nd5", "mt-Nd6", "mt-Cytb", "AC125149.3", "AC125149.5", "AC125149.1",
"AC125149.2", "AC125149.4", "AC133103.4", "AC133103.6", "AC133103.7", "AC133103.5",
"AC133103.1", "AC133103.3", "AC168977.2", "AC168977.1", "CAAA01118383.1",
"Vmn1r186", "AC102264.1", "AC132444.1", "AC132444.3", "AC132444.5", "AC132444.4",
"AC132444.2", "Csprs", "AC132444.6", "Vamp7", "CAAA01165726.1", "Spry3", "Tmlhe",
"Ccl21b.1", "AC087559.3", "Fam205a4.1", "AC087559.2", "Ccl21c", "AC140325.2",
"AC140325.1", "Gm3286.1", "AC140325.3", "AC140325.4", "Vmn2r122", "Il11ra2.1",
"Ccl27a.1", "CR974586.2", "Ccl21c.1", "CR974586.3", "CR974586.5", "CR974586.4",
"4933409K07Rik", "CR974586.1", "Ccl27a.2", "Il11ra2.2", "Ccl19.1", "Ccl21a.1",
"Gm10931", "CT868723.1", "CAAA01147332.1", "AC164084.2", "AC164084.3",
"AC164084.1", "AC165294.1", "AC165294.2", "AC165294.3", "AC125178.1", "AC125178.3",
"AC125178.2", "AC123873.3", "AC123873.1", "AC123873.2", "AC126035.1", "Gm16367",
"AC163611.1", "AC163611.2", "AC140365.1", "AC124606.2", "AC124606.1", "AC133095.2",
"AC133095.1", "AC234645.1", or "AC149090.1", not "counts".
ℹ Did you mean "Clint1"?

Backtrace:
▆

1. ├─Seurat::PercentageFeatureSet(wt, pattern = "^mt-")
2. │ ├─features %||% ...
3. │ ├─base::grep(...)
4. │ │ └─base::is.factor(x)
5. │ ├─base::rownames(x = object[[assay]][layer])
6. │ ├─object[[assay]][layer]
7. │ └─SeuratObject:::[.Assay(object[[assay]], layer)
8. │ ├─SeuratObject::LayerData(...)
9. │ └─SeuratObject:::LayerData.Assay(features = i)
10. │ └─rlang::arg_match(...)
11. │ └─rlang:::arg_match_multi(arg, values, error_arg, error_call)
12. │ └─rlang:::map_chr(arg, ~arg_match0(.x, values, error_arg, error_call = error_call))
13. │ └─rlang:::.rlang_purrr_map_mold(.x, .f, character(1), ...)
14. │ └─base::vapply(.x, .f, .mold, ..., USE.NAMES = FALSE)
15. │ └─rlang (local) FUN(X[[i]], ...)
16. │ └─rlang::arg_match0(.x, values, error_arg, error_call = error_call)
17. └─rlang:::stop_arg_match(w, values = x, error_arg = y, error_call = z)
18. └─rlang::abort(msg, call = error_call, arg = error_arg)

Thank you,
Iva

[samuel-marsh]

Hi,

Can you please post the output of sessionInfo()?

Thanks,
Sam

[iva302]

sessionInfo()
R version 4.1.2 (2021-11-01)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.2 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 LC_TIME=en_US.UTF-8
[4] LC_COLLATE=en_US.UTF-8 LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C LC_ADDRESS=C
[10] LC_TELEPHONE=C LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

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

other attached packages:
[1] patchwork_1.1.3 ggplot2_3.4.4 EnsDb.Mmusculus.v79_2.99.0
[4] ensembldb_2.18.4 AnnotationFilter_1.18.0 GenomicFeatures_1.46.5
[7] AnnotationDbi_1.56.2 Biobase_2.54.0 GenomicRanges_1.46.1
[10] GenomeInfoDb_1.30.1 IRanges_2.28.0 S4Vectors_0.32.4
[13] BiocGenerics_0.40.0 Seurat_5.0.1 SeuratObject_5.0.1
[16] sp_2.1-2 Signac_1.12.0 dplyr_1.1.4
[19] magrittr_2.0.3

loaded via a namespace (and not attached):
[1] spam_2.10-0 fastmatch_1.1-4
[3] BiocFileCache_2.2.1 plyr_1.8.9
[5] igraph_1.5.1 lazyeval_0.2.2
[7] splines_4.1.2 RcppHNSW_0.5.0
[9] BiocParallel_1.28.3 listenv_0.9.0
[11] scattermore_1.2 digest_0.6.33
[13] htmltools_0.5.7 fansi_1.0.6
[15] memoise_2.0.1 tensor_1.5
[17] cluster_2.1.6 ROCR_1.0-11
[19] globals_0.16.2 Biostrings_2.62.0
[21] matrixStats_1.1.0 spatstat.sparse_3.0-3
[23] prettyunits_1.2.0 colorspace_2.1-0
[25] rappdirs_0.3.3 blob_1.2.4
[27] ggrepel_0.9.4 crayon_1.5.2
[29] RCurl_1.98-1.13 jsonlite_1.8.8
[31] progressr_0.14.0 spatstat.data_3.0-3
[33] survival_3.5-7 zoo_1.8-12
[35] glue_1.7.0 polyclip_1.10-6
[37] gtable_0.3.4 zlibbioc_1.40.0
[39] XVector_0.34.0 leiden_0.4.3.1
[41] DelayedArray_0.20.0 future.apply_1.11.0
[43] abind_1.4-5 scales_1.3.0
[45] DBI_1.2.1 spatstat.random_3.2-2
[47] miniUI_0.1.1.1 Rcpp_1.0.11
[49] progress_1.2.3 viridisLite_0.4.2
[51] xtable_1.8-4 reticulate_1.34.0
[53] bit_4.0.5 dotCall64_1.1-1
[55] htmlwidgets_1.6.4 httr_1.4.7
[57] RColorBrewer_1.1-3 ellipsis_0.3.2
[59] ica_1.0-3 XML_3.99-0.16
[61] pkgconfig_2.0.3 dbplyr_2.3.4
[63] uwot_0.1.16 deldir_2.0-2
[65] utf8_1.2.4 tidyselect_1.2.1
[67] rlang_1.1.3 reshape2_1.4.4
[69] later_1.3.2 munsell_0.5.0
[71] tools_4.1.2 cachem_1.0.8
[73] cli_3.6.2 generics_0.1.3
[75] RSQLite_2.3.4 ggridges_0.5.4
[77] stringr_1.5.1 fastmap_1.1.1
[79] yaml_2.3.7 goftest_1.2-3
[81] bit64_4.0.5 fitdistrplus_1.1-11
[83] purrr_1.0.2 RANN_2.6.1
[85] KEGGREST_1.34.0 pbapply_1.7-2
[87] future_1.33.0 nlme_3.1-164
[89] mime_0.12 RcppRoll_0.3.0
[91] xml2_1.3.6 biomaRt_2.50.3
[93] compiler_4.1.2 rstudioapi_0.15.0
[95] filelock_1.0.2 curl_5.2.0
[97] plotly_4.10.3 png_0.1-8
[99] spatstat.utils_3.0-4 tibble_3.2.1
[101] stringi_1.8.3 RSpectra_0.16-1
[103] lattice_0.22-5 ProtGenerics_1.26.0
[105] Matrix_1.6-4 vctrs_0.6.5
[107] pillar_1.9.0 lifecycle_1.0.4
[109] spatstat.geom_3.2-7 lmtest_0.9-40
[111] RcppAnnoy_0.0.21 data.table_1.15.4
[113] cowplot_1.1.1 bitops_1.0-7
[115] irlba_2.3.5.1 rtracklayer_1.54.0
[117] httpuv_1.6.13 BiocIO_1.4.0
[119] R6_2.5.1 promises_1.2.1
[121] KernSmooth_2.23-20 gridExtra_2.3
[123] parallelly_1.36.0 codetools_0.2-18
[125] fastDummies_1.7.3 MASS_7.3-60
[127] SummarizedExperiment_1.24.0 rjson_0.2.21
[129] withr_3.0.0 GenomicAlignments_1.30.0
[131] sctransform_0.4.1 Rsamtools_2.10.0
[133] GenomeInfoDbData_1.2.7 hms_1.1.3
[135] parallel_4.1.2 grid_4.1.2
[137] tidyr_1.3.1 MatrixGenerics_1.6.0
[139] Rtsne_0.17 spatstat.explore_3.2-5
[141] shiny_1.8.0 restfulr_0.0.15

[samuel-marsh]

Hi @iva302,

If you update Seurat to current cran release (5.0.3) do you get the same error?

Best,
Sam

[iva302]

Hello,
I uninstalled older Seurat version and installed 5.0.3 version and I am still getting the same error-

sessionInfo()
R version 4.1.2 (2021-11-01)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.2 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 LC_TIME=en_US.UTF-8
[4] LC_COLLATE=en_US.UTF-8 LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C LC_ADDRESS=C
[10] LC_TELEPHONE=C LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

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

other attached packages:
[1] patchwork_1.2.0 ggplot2_3.5.1 EnsDb.Mmusculus.v79_2.99.0
[4] ensembldb_2.18.4 AnnotationFilter_1.18.0 GenomicFeatures_1.46.5
[7] AnnotationDbi_1.56.2 Biobase_2.54.0 GenomicRanges_1.46.1
[10] GenomeInfoDb_1.30.1 IRanges_2.28.0 S4Vectors_0.32.4
[13] BiocGenerics_0.40.0 Seurat_5.0.3 SeuratObject_5.0.1
[16] sp_2.1-3 Signac_1.12.0 dplyr_1.1.4
[19] magrittr_2.0.3

loaded via a namespace (and not attached):
[1] utf8_1.2.4 spatstat.explore_3.2-7
[3] reticulate_1.36.1 tidyselect_1.2.1
[5] RSQLite_2.3.4 htmlwidgets_1.6.4
[7] grid_4.1.2 BiocParallel_1.28.3
[9] Rtsne_0.17 munsell_0.5.1
[11] codetools_0.2-18 ica_1.0-3
[13] future_1.33.2 miniUI_0.1.1.1
[15] withr_3.0.0 spatstat.random_3.2-3
[17] colorspace_2.1-0 progressr_0.14.0
[19] filelock_1.0.2 knitr_1.46
[21] rstudioapi_0.15.0 ROCR_1.0-11
[23] tensor_1.5 listenv_0.9.1
[25] MatrixGenerics_1.6.0 GenomeInfoDbData_1.2.7
[27] polyclip_1.10-6 bit64_4.0.5
[29] parallelly_1.37.1 vctrs_0.6.5
[31] generics_0.1.3 xfun_0.43
[33] biovizBase_1.42.0 BiocFileCache_2.2.1
[35] R6_2.5.1 hdf5r_1.3.8
[37] bitops_1.0-7 spatstat.utils_3.0-4
[39] cachem_1.0.8 DelayedArray_0.20.0
[41] promises_1.3.0 BiocIO_1.4.0
[43] scales_1.3.0 nnet_7.3-19
[45] gtable_0.3.5 globals_0.16.3
[47] processx_3.8.2 goftest_1.2-3
[49] spam_2.10-0 rlang_1.1.3
[51] RcppRoll_0.3.0 splines_4.1.2
[53] rtracklayer_1.54.0 lazyeval_0.2.2
[55] dichromat_2.0-0.1 checkmate_2.3.1
[57] spatstat.geom_3.2-9 yaml_2.3.8
[59] reshape2_1.4.4 abind_1.4-5
[61] backports_1.4.1 httpuv_1.6.15
[63] Hmisc_5.1-1 tools_4.1.2
[65] RColorBrewer_1.1-3 ggridges_0.5.6
[67] Rcpp_1.0.12 plyr_1.8.9
[69] base64enc_0.1-3 progress_1.2.3
[71] zlibbioc_1.40.0 purrr_1.0.2
[73] RCurl_1.98-1.13 ps_1.7.5
[75] prettyunits_1.2.0 rpart_4.1.23
[77] deldir_2.0-4 pbapply_1.7-2
[79] cowplot_1.1.3 zoo_1.8-12
[81] SummarizedExperiment_1.24.0 ggrepel_0.9.5
[83] cluster_2.1.6 data.table_1.15.4
[85] RSpectra_0.16-1 scattermore_1.2
[87] lmtest_0.9-40 RANN_2.6.1
[89] ProtGenerics_1.26.0 fitdistrplus_1.1-11
[91] matrixStats_1.3.0 evaluate_0.23
[93] hms_1.1.3 mime_0.12
[95] xtable_1.8-4 XML_3.99-0.16
[97] fastDummies_1.7.3 gridExtra_2.3
[99] compiler_4.1.2 biomaRt_2.50.3
[101] tibble_3.2.1 KernSmooth_2.23-20
[103] crayon_1.5.2 htmltools_0.5.8.1
[105] later_1.3.2 Formula_1.2-5
[107] tidyr_1.3.1 DBI_1.2.1
[109] dbplyr_2.3.4 MASS_7.3-60
[111] rappdirs_0.3.3 Matrix_1.6-4
[113] cli_3.6.2 parallel_4.1.2
[115] dotCall64_1.1-1 igraph_2.0.3
[117] pkgconfig_2.0.3 GenomicAlignments_1.30.0
[119] foreign_0.8-86 plotly_4.10.4
[121] spatstat.sparse_3.0-3 xml2_1.3.6
[123] XVector_0.34.0 VariantAnnotation_1.40.0
[125] stringr_1.5.1 callr_3.7.3
[127] digest_0.6.35 sctransform_0.4.1
[129] RcppAnnoy_0.0.22 spatstat.data_3.0-4
[131] Biostrings_2.62.0 rmarkdown_2.26
[133] leiden_0.4.3.1 fastmatch_1.1-4
[135] htmlTable_2.4.2 uwot_0.2.2
[137] restfulr_0.0.15 curl_5.2.1
[139] shiny_1.8.1.1 Rsamtools_2.10.0
[141] rjson_0.2.21 lifecycle_1.0.4
[143] nlme_3.1-164 jsonlite_1.8.8
[145] BSgenome_1.62.0 viridisLite_0.4.2
[147] fansi_1.0.6 pillar_1.9.0
[149] lattice_0.22-5 KEGGREST_1.34.0
[151] fastmap_1.1.1 httr_1.4.7
[153] pkgbuild_1.4.2 survival_3.5-7
[155] glue_1.7.0 remotes_2.4.2.1
[157] png_0.1-8 bit_4.0.5
[159] stringi_1.8.3 blob_1.2.4
[161] RcppHNSW_0.6.0 memoise_2.0.1
[163] irlba_2.3.5.1 future.apply_1.11.2

[igrabski]

Hi, can you show what the output of your object wt looks like, and what your input counts rna_counts looks like?

[iva302]

Hello,
This is the output of my object wt-

wt
An object of class Seurat
32285 features across 20000 samples within 1 assay
Active assay: RNA (32285 features, 0 variable features)
2 layers present: counts, data

Is this what you mean by rna_counts?

wt$nCount_RNA
AAACAGCCAACTAGGG-1 AAACAGCCAAGGTAAC-1 AAACAGCCAAGTGAAC-1 AAACAGCCAATAACGA-1
3507 2507 2324 2663
AAACAGCCACAAAGCG-1 AAACAGCCACAACCTA-1 AAACAGCCACATTAAC-1 AAACAGCCACTAGCGT-1
1380 4056 2564 1285
AAACAGCCAGCAATAA-1 AAACAGCCAGCATTAT-1 AAACAGCCAGGAACAT-1 AAACAGCCAGGCATCT-1
1992 1729 783 974
AAACAGCCAGTTGCGT-1 AAACAGCCATCAGCAC-1 AAACAGCCATTCAGCA-1 AAACAGCCATTCCTGT-1
1183 804 1432 2074

Thank you,
Iva

[igrabski]

Thanks! And sorry, by rna_counts, I am referring to your object called rna_counts (from this line of code, rna_counts <- inputdata.10x$Gene Expression)

[iva302]

Hello,
This is output of rna_counts-

rna_counts
32285 x 20000 sparse Matrix of class "dgCMatrix"
[[ suppressing 35 column names 'AAACAGCCAACTAGGG-1', 'AAACAGCCAAGGTAAC-1', 'AAACAGCCAAGTGAAC-1' ... ]]
[[ suppressing 35 column names 'AAACAGCCAACTAGGG-1', 'AAACAGCCAAGGTAAC-1', 'AAACAGCCAAGTGAAC-1' ... ]]

Xkr4 . . . . . . . 3 . . . . . . 1 . . . . 1 1 2 . . . . . . . . . . . . . ......
Gm1992 . . . . . . . 1 . . . . . . . . . . . . . . . 1 . . . . . . . . . . . ......
Gm19938 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Gm37381 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Rp1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Sox17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Gm37587 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Gm37323 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Mrpl15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Lypla1 . 2 . 2 . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Tcea1 . 2 1 . . . . . . 1 1 . . 1 . . 1 1 . 4 1 . 1 4 . 1 1 . . . . . . 2 . ......
Rgs20 . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Gm16041 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Atp6v1h 1 . . . . . . . . . . . . 1 . 1 1 . . 1 . . . . . . . . . . . . . 2 . ......

..............................
........suppressing 19965 columns and 32257 rows in show(); maybe adjust options(max.print=, width=)
..............................
[[ suppressing 35 column names 'AAACAGCCAACTAGGG-1', 'AAACAGCCAAGGTAAC-1', 'AAACAGCCAAGTGAAC-1' ... ]]

AC123873.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC123873.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC123873.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC126035.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
Gm16367 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC163611.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC163611.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC140365.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC124606.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC124606.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC133095.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC133095.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC234645.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......
AC149090.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......

Thank you,
Iva