reference for RunAzimuth()

[cathalgking]

I am running seurat version 5 and Azimuth 0.4.6 and RunAzimuth() cannot seem to find my reference. I have tried running it with various different references. 1. When I download a reference (ref.rds and idx.annoy files) from zenodo and place in a folder on my computer I get: Error in RunAzimuth.Seurat( ... Could not find a reference for /users/ .../ref.rds 2. When I run with the built in bone marrow ref "bmcite" (which says it is installed when I run InstalledData()) I get: Error in LoadReference(file.path(system.file(package = ds), type)) : Missing the following files from the directory provided: ref.Rdsidx.annoy ran seu_test_Az <- RunAzimuth(seu, reference = "bmcite") 3. When I run with 'pbmcref' it starts to process but then gives the error: Error in validObject(object = object) : invalid class “DimReduc” object: colnames for ‘feature.loadings’ must start with reduction key (refdr_)

Lastly, I would also like to use a different reference for annotation (Tabula sapiens bone marrow converted to .rds format) but I do not have the .annoy file - how can I make one?

[MWRR]

@cathalgking Have you solved the issue, I got the same error When I run with 'pbmcref' it starts to process but then gives the error: Error in validObject(object = object) : invalid class “DimReduc” object: colnames for ‘feature.loadings’ must start with reduction key (refdr_) any help?

[Gesmira]

Hi both, please install the v5 version of Azimuth for use with Seurat v5.

remotes::install_github("satijalab/seurat-data", "seurat5", quiet = TRUE)
remotes::install_github("satijalab/azimuth", "seurat5", quiet = TRUE)

[Gesmira]

In terms of the second question, you can make the annoy index file with the following code: SaveAnnoyIndex(object = obj.azimuth[["refdr.annoy.neighbors"]], file = "reference/idx.annoy")

[ERIGR]

Hi, I have the same issue when using Seurat5 with the fetus reference. Firstly I try directly to map my query to the previously downloaded fetal reference. query <- RunAzimuth(query,reference = "/WORKDIR/mnt/beegfs/userdata/e_robert/single_cell_reference_dataset/fetusref.SeuratData/inst/azimuth/") and get the following error message :

Warning: Overwriting miscellanous data for model Warning: Adding a dimensional reduction (refUMAP) without the associated assay being present Warning: Adding a dimensional reduction (refUMAP) without the associated assay being present detected inputs from HUMAN with id type Gene.name reference rownames detected HUMAN with id type Gene.name Running SCTransform on assay: RNA Using reference SCTModel to calculate pearson residuals Determine variable features Setting minvariance to: -Inf Calculating residuals of type pearson for 2031 genes |======================================================================| 100% |======================================================================| 100% Set default assay to refAssay Normalizing query using reference SCT model Error in validObject(object = object) : invalid class "DimReduc" object: colnames for 'feature.loadings' must start with reduction key (refdr) Calls: RunAzimuth ... RenameCells.Seurat -> RenameCells -> RenameCells.DimReduc -> validObject

Then I try to save the annoy Index as you mentionned it above : SaveAnnoyIndex(ref@neighbors[["refdr.annoy.neighbors"]],"/path/fetal_reference/idx.annoy")

and get the following mistake :
Error in SaveAnnoyIndex(ref@neighbors[["refdr.annoy.neighbors"]], "path/fetal_reference/idx.annoy") : Index for provided Neighbor object is NULL I get the same running exactly the command you mentionned : SaveAnnoyIndex(object = ref[["refdr.annoy.neighbors"]], file = "reference/idx.annoy")

installed.packages()[,which(colnames(installed.packages())=="Version")]
AnnotationDbi AnnotationFilter "1.60.2" "1.22.0" Azimuth BH "0.4.6" "1.81.0-1" BSgenome BSgenome.Hsapiens.UCSC.hg38 "1.66.3" "1.4.5" Biobase BiocFileCache "2.58.0" "2.6.1" BiocGenerics BiocIO "0.44.0" "1.8.0" BiocManager BiocParallel "1.30.20" "1.32.6" BiocVersion Biostrings "3.16.0" "2.66.0" CNEr DBI "1.34.0" "1.1.3" DT DelayedArray "0.28" "0.24.0" DelayedMatrixStats DirichletMultinomial "1.20.0" "1.40.0" EnsDb.Hsapiens.v86 FNN "2.99.0" "1.1.3.2" GO.db GenomeInfoDb "3.16.0" "1.34.9" GenomeInfoDbData GenomicAlignments "1.2.9" "1.34.1" GenomicFeatures GenomicRanges "1.50.4" "1.50.2" HDF5Array IRanges "1.26.0" "2.32.0" JASPAR2020 KEGGREST "0.99.10" "1.38.0" KernSmooth MASS "2.23-21" "7.3-60" Matrix MatrixGenerics "1.5-4.1" "1.10.0" MatrixModels ProtGenerics "0.5-1" "1.30.0" R.methodsS3 R.oo "1.8.2" "1.25.0" R.utils R6 "2.12.2" "2.5.1" RANN RColorBrewer "2.6.1" "1.1-3" RCurl ROCR "1.98-1.12" "1.0-11" RSQLite RSpectra "2.3.1" "0.16-1" Rcpp RcppAnnoy "1.0.10" "0.0.20" RcppArmadillo RcppEigen "0.12.2.0.0" "0.3.3.9.3" RcppHNSW RcppProgress "0.4.1" "0.4.2" RcppRoll RcppTOML "0.3.0" "0.2.2" Rhdf5lib Rhtslib "1.20.0" "2.0.0" Rsamtools Rtsne "2.14.0" "0.16" S4Vectors Seurat "0.36.2" "4.9.9.9045" SeuratData SeuratDisk "0.2.2" "0.0.0.9020" SeuratObject SeuratWrappers "4.9.9.9084" "0.3.19" Signac SingleCellExperiment "1.9.0.9000" "1.20.1" SparseM SummarizedExperiment "1.81" "1.28.0" TFBSTools TFMPvalue "1.36.0" "0.0.9" XML XVector "3.99-0.14" "0.38.0" abind annotate "1.4-5" "1.76.0" askpass assertthat "1.1" "0.2.1" backports base64enc "1.4.1" "0.1-3" basilisk basilisk.utils "1.10.2" "1.10.0" beachmat biomaRt "2.14.2" "2.54.1" bit bit64 "4.0.5" "4.0.5" bitops blob "1.0-7" "1.2.4" boot brew "1.3-28.1" "1.0-8" brio broom "1.1.3" "1.0.4" bslib caTools "0.4.2" "1.18.2" cachem callr "1.0.8" "3.7.3" car carData "3.1-2" "3.0-5" cellranger chameleon "1.1.0" "0.2-3" cli clipr "3.6.1" "0.8.0" clue cluster "0.3-64" "2.1.4" codetools colorspace "0.2-19" "2.1-0" commonmark corrplot "1.9.0" "0.92" covr cowplot "3.6.2" "1.1.1" cpp11 crayon "0.4.3" "1.5.2" credentials crosstalk "1.3.2" "1.2.0" curl data.table "5.0.0" "1.14.8" data.tree dbplyr "1.0.0" "2.3.2" deldir densvis "1.0-9" "1.8.3" desc devtools "1.4.2" "2.4.5" diffobj diffviewer "0.3.5" "0.1.1" digest dir.expiry "0.6.31" "1.6.0" doParallel dotCall64 "1.0.17" "1.0-2" downlit dplyr "0.4.2" "1.1.2" dqrng ellipsis "0.3.0" "0.3.2" ensembldb evaluate "2.22.0" "0.21" fansi farver "1.0.4" "2.1.1" fastDummies fastmap "1.6.3" "1.1.1" fastmatch fetusref.SeuratData "1.1-3" "1.0.0" filelock fitdistrplus "1.0.2" "1.1-11" fontawesome foreach "0.5.1" "1.5.2" formatR fs "1.14" "1.6.2" futile.logger futile.options "1.4.3" "1.0.1" future future.apply "1.32.0" "1.11.0" gargle generics "1.4.0" "0.1.3" gert getopt "1.9.2" "1.20.3" ggplot2 ggpubr "3.4.2" "0.6.0" ggrepel ggridges "0.9.3" "0.5.4" ggsci ggsignif "3.0.0" "0.6.4" gh gitcreds "1.4.0" "0.1.2" glmGamPoi globals "1.10.2" "0.16.2" glue goftest "1.6.2" "1.2-3" googledrive 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"4.10.1" "1.8.8" png polyclip "0.1-8" "1.10-4" polynom poweRlaw "1.4-1" "0.70.6" pracma praise "2.4.2" "1.0.0" presto prettyunits "1.0.0" "1.1.1" processx profvis "3.8.1" "0.3.8" progress progressr "1.2.2" "0.13.0" promises ps "1.2.0.1" "1.7.5" purrr quantreg "1.0.1" "5.95" ragg rappdirs "1.2.5" "0.3.3" rcmdcheck readr "1.4.0" "2.1.4" readxl rematch "1.4.2" "1.0.1" rematch2 remotes "2.1.2" "2.4.2" reshape2 restfulr "1.4.4" "0.0.15" reticulate rex "1.28" "1.2.1" rhdf5 rhdf5filters "2.42.1" "1.10.1" rjson rlang "0.2.21" "1.1.1" rmarkdown roxygen2 "2.21" "7.2.3" rpart rprojroot "4.1.19" "2.0.3" rstatix rstudioapi "0.7.2" "0.14" rsvd rtracklayer "1.0.5" "1.58.0" rversions sass "2.1.2" "0.4.6" scMRMA scales "1.0" "1.2.1" scattermore sctransform "1.1" "0.3.5" seqLogo sessioninfo "1.64.0" "1.2.2" shiny shinyBS "1.7.4" "0.61.1" shinydashboard shinyjs "0.7.2" "2.1.0" sitmo snow "2.0.2" "0.4-4" sourcetools sp "0.1.7-1" "1.6-0" spam sparseMatrixStats "2.9-1" "1.10.0" spatstat spatstat.data "1.64-2" "3.0-1" spatstat.explore spatstat.geom "3.2-1" "3.2-1" spatstat.random spatstat.sparse "3.1-5" "3.0-1" spatstat.utils stringi "3.0-3" "1.7.12" stringr survival "1.5.0" "3.5-5" sys systemfonts "3.4.2" "1.0.4" tensor testthat "1.5" "3.1.8" textshaping tibble "0.3.6" "3.2.1" tidyr tidyselect "1.3.0" "1.2.0" tinytex tzdb "0.45" "0.4.0" umap urlchecker "0.2.10.0" "1.0.1" usethis utf8 "2.1.6" "1.2.3" uuid uwot "1.1-0" "0.1.14" vctrs viridisLite "0.6.2" "0.4.2" vroom waldo "1.6.3" "0.5.1" whisker withr "0.4.1" "2.5.0" xfun xml2 "0.39" "1.3.4" xopen xtable "1.0.0" "1.8-4" yaml zip "2.3.7" "2.3.0" zlibbioc zoo "1.44.0" "1.8-12" base compiler "4.2.2" "4.2.2" datasets grDevices "4.2.2" "4.2.2" graphics grid "4.2.2" "4.2.2" methods parallel "4.2.2" "4.2.2" splines stats "4.2.2" "4.2.2" stats4 tcltk "4.2.2" "4.2.2" tools utils "4.2.2" "4.2.2"

[Gesmira]

Hi @ERIGR, When you say you ran this with Seurat5, did you also restart your session, install and load the new version of Azimuth I mentioned above?

In terms of the second problem you mentioned, if you have saved and reloaded the object, the annoy index will disappear. Please rerun AzimuthReference() and then immediately run the SaveAnnoyIndex() function.

[ERIGR]

Hi @Gesmira, I do analysis with a singularity image so actually I have difficulty to install the seuratdata package that is why I used the files provided by zenodo (rds and annoy file) for the fetus reference. The version of azimuth is actually the good version (seurat5). I use the following command to install it : R -e 'install.packages("withr")' R -e 'remotes::install_github("satijalab/seurat", "seurat5", quiet = TRUE)' R -e 'remotes::install_github("satijalab/seurat-data", "seurat5", quiet = TRUE)' R -e 'remotes::install_github("satijalab/azimuth", "seurat5", quiet = TRUE)' R -e 'remotes::install_github("satijalab/seurat-wrappers", "seurat5", quiet = TRUE)' R -e 'remotes::install_github("stuart-lab/signac", "seurat5", quiet = TRUE)'

Actually, I'm wondering if there is a way to retrieve the fetus reference suitable with seurat5 directly without having to use SeuratData. Eventually can you send it to me directly ? Thanks

[cathalgking]

@Gesmira I cannot even get Azimuth (v5) to install. Dont usually get warning messages like this:

Installing 7 packages: sys, deldir, parallelly, RcppArmadillo, knitr, igraph, scattermore Warning message: In i.p(...) : installation of package ‘/var/folders/k7/tgj9fjld27ng9fryktpj53108z9y55/T//RtmpYppHLC/file2ceb742888dd/Azimuth_0.4.6.9004.tar.gz’ had non-zero exit status

[Gesmira]

@cathalgking are you using quiet = TRUE in your installation? If you could rerun with quiet = FALSE we could see what is going wrong. I'd recommend opening a new issue as it is unrelated to the current errors.

@ERIGR, I am unable to reproduce your issue with all of the updated versions. If you'd like to provide me with your email, I can send you the updated fetus reference object.

[jessievanbuggenum]

Hi @cathalgking and @MWRR , is your issue solved?

Yesterday I ran for the first time RunAzimuth(obj, reference = "data/references/mouse_motor_cortex/",assay = "RNA",umap.name = "umap.ref",verbose = TRUE) which worked. Today I ran the exact same dataset and function again, and I got the same error:

Error in validObject(object = object) : invalid class “DimReduc” object: colnames for ‘feature.loadings’ must start with reduction key (refdr_)

This error is proceeded by:

Warning: Overwriting miscellanous data for model Warning: Adding a dimensional reduction (refUMAP) without the associated assay being present Warning: Adding a dimensional reduction (refUMAP) without the associated assay being present detected inputs from MOUSE with id type Gene.name reference rownames detected MOUSE with id type Gene.name Running SCTransform on assay: RNA Using reference SCTModel to calculate pearson residuals Determine variable features Setting min_variance to: -Inf Calculating residuals of type pearson for 2935 genes |===========================================================================================================================================================| 100% |===========================================================================================================================================================| 100% Set default assay to refAssay Normalizing query using reference SCT model

I tried to reinstall packages, restart Rsession etc, and also to 'clean' my dataset and run new dim reduction, but maybe in the wrong order or so. Does anyone know what the problem is?

[Gesmira]

@jessievanbuggenum Can you share the output of sessionInfo()?

[jessievanbuggenum]

R version 4.3.0 (2023-04-21) Platform: aarch64-apple-darwin20 (64-bit) Running under: macOS Ventura 13.4

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

other attached packages: [1] Seurat_4.9.9.9045 SeuratObject_4.9.9.9084 sp_1.6-1 tidyr_1.3.0 SingleCellExperiment_1.22.0 [6] SummarizedExperiment_1.30.1 Biobase_2.60.0 GenomicRanges_1.52.0 GenomeInfoDb_1.36.0 IRanges_2.34.0
[11] S4Vectors_0.38.1 BiocGenerics_0.46.0 MatrixGenerics_1.12.0 matrixStats_1.0.0 future_1.32.0
[16] tibble_3.2.1 openxlsx_4.2.5.2 dplyr_1.1.2 ggplot2_3.4.2 patchwork_1.1.2
[21] SeuratData_0.2.2 Azimuth_0.4.6 shinyBS_0.61.1

loaded via a namespace (and not attached): [1] RcppAnnoy_0.0.20 splines_4.3.0 later_1.3.1 BiocIO_1.10.0 bitops_1.0-7 cellranger_1.1.0
[7] polyclip_1.10-4 XML_3.99-0.14 fastDummies_1.6.3 lifecycle_1.0.3 scDblFinder_1.14.0 edgeR_3.42.2
[13] hdf5r_1.3.8 globals_0.16.2 lattice_0.21-8 MASS_7.3-58.4 magrittr_2.0.3 limma_3.56.1
[19] plotly_4.10.1 yaml_2.3.7 metapod_1.8.0 httpuv_1.6.11 sctransform_0.3.5 spam_2.9-1
[25] zip_2.3.0 spatstat.sparse_3.0-1 reticulate_1.28 cowplot_1.1.1 pbapply_1.7-0 RColorBrewer_1.1-3
[31] abind_1.4-5 zlibbioc_1.46.0 Rtsne_0.16 presto_1.0.0 purrr_1.0.1 RCurl_1.98-1.12
[37] rappdirs_0.3.3 GenomeInfoDbData_1.2.10 ggrepel_0.9.3 irlba_2.3.5.1 spatstat.utils_3.0-3 listenv_0.9.0
[43] goftest_1.2-3 RSpectra_0.16-1 spatstat.random_3.1-5 dqrng_0.3.0 fitdistrplus_1.1-11 parallelly_1.36.0
[49] DelayedMatrixStats_1.22.0 leiden_0.4.3 codetools_0.2-19 DelayedArray_0.26.3 DT_0.28 scuttle_1.10.1
[55] tidyselect_1.2.0 farver_2.1.1 ScaledMatrix_1.8.1 viridis_0.6.3 spatstat.explore_3.2-1 googledrive_2.1.0
[61] GenomicAlignments_1.36.0 jsonlite_1.8.4 BiocNeighbors_1.18.0 ellipsis_0.3.2 progressr_0.13.0 ggridges_0.5.4
[67] survival_3.5-5 scater_1.28.0 tools_4.3.0 ica_1.0-3 Rcpp_1.0.10 glue_1.6.2
[73] gridExtra_2.3 shinydashboard_0.7.2 withr_2.5.0 BiocManager_1.30.20 fastmap_1.1.1 bluster_1.10.0
[79] fansi_1.0.4 shinyjs_2.1.0 digest_0.6.31 rsvd_1.0.5 R6_2.5.1 mime_0.12
[85] colorspace_2.1-0 scattermore_1.1 tensor_1.5 spatstat.data_3.0-1 googlesheets4_1.1.0 utf8_1.2.3
[91] generics_0.1.3 renv_0.17.3 data.table_1.14.8 rtracklayer_1.60.0 httr_1.4.6 htmlwidgets_1.6.2
[97] S4Arrays_1.0.4 uwot_0.1.14 pkgconfig_2.0.3 gtable_0.3.3 lmtest_0.9-40 XVector_0.40.0
[103] htmltools_0.5.5 dotCall64_1.0-2 scales_1.2.1 png_0.1-8 harmony_0.1.1 scran_1.28.1
[109] rstudioapi_0.14 reshape2_1.4.4 rjson_0.2.21 curl_5.0.0 nlme_3.1-162 zoo_1.8-12
[115] stringr_1.5.0 KernSmooth_2.23-20 parallel_4.3.0 miniUI_0.1.1.1 vipor_0.4.5 restfulr_0.0.15
[121] pillar_1.9.0 grid_4.3.0 vctrs_0.6.2 RANN_2.6.1 promises_1.2.0.1 BiocSingular_1.16.0
[127] beachmat_2.16.0 SeuratDisk_0.0.0.9020 xtable_1.8-4 cluster_2.1.4 beeswarm_0.4.0 cli_3.6.1
[133] locfit_1.5-9.7 compiler_4.3.0 Rsamtools_2.16.0 rlang_1.1.1 crayon_1.5.2 future.apply_1.11.0
[139] labeling_0.4.2 fs_1.6.2 plyr_1.8.8 ggbeeswarm_0.7.2 stringi_1.7.12 deldir_1.0-9
[145] viridisLite_0.4.2 BiocParallel_1.34.2 munsell_0.5.0 Biostrings_2.68.1 lazyeval_0.2.2 spatstat.geom_3.2-1
[151] Matrix_1.5-4 RcppHNSW_0.4.1 bit64_4.0.5 sparseMatrixStats_1.12.0 statmod_1.5.0 shiny_1.7.4
[157] ROCR_1.0-11 gargle_1.4.0 igraph_1.4.3 bit_4.0.5 xgboost_1.7.5.1

[jessievanbuggenum]

Hi @Gesmira, Previous comment session info when I get the error.

Small update: In a new and clean Renvironment, I installed azimuth anew and only loaded azimuth in the session. I read the rds file and called RunAzimuth. This time it does run well, but funny enough, the Azimuth version itself is the same. So maybe some conflict with loading other packages after loading Azimuth?

Sessioninfo() R version 4.3.0 (2023-04-21) Platform: aarch64-apple-darwin20 (64-bit) Running under: macOS Ventura 13.4

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

other attached packages: [1] Azimuth_0.4.6 shinyBS_0.61.1

loaded via a namespace (and not attached): [1] RColorBrewer_1.1-3 rstudioapi_0.14 jsonlite_1.8.4 magrittr_2.0.3 spatstat.utils_3.0-3 fs_1.6.2
[7] vctrs_0.6.2 ROCR_1.0-11 spatstat.explore_3.2-1 htmltools_0.5.5 curl_5.0.0 cellranger_1.1.0
[13] googlesheets4_1.1.0 sctransform_0.3.5 parallelly_1.36.0 KernSmooth_2.23-20 htmlwidgets_1.6.2 ica_1.0-3
[19] plyr_1.8.8 plotly_4.10.1 zoo_1.8-12 igraph_1.4.3 mime_0.12 lifecycle_1.0.3
[25] pkgconfig_2.0.3 Matrix_1.5-4 R6_2.5.1 fastmap_1.1.1 fitdistrplus_1.1-11 future_1.32.0
[31] shiny_1.7.4 digest_0.6.31 colorspace_2.1-0 patchwork_1.1.2 SeuratDisk_0.0.0.9020 Seurat_4.3.0
[37] tensor_1.5 irlba_2.3.5.1 progressr_0.13.0 fansi_1.0.4 spatstat.sparse_3.0-1 httr_1.4.6
[43] polyclip_1.10-4 abind_1.4-5 compiler_4.3.0 gargle_1.4.0 bit64_4.0.5 withr_2.5.0
[49] MASS_7.3-58.4 rappdirs_0.3.3 SeuratData_0.2.2 tools_4.3.0 lmtest_0.9-40 googledrive_2.1.0
[55] httpuv_1.6.11 future.apply_1.11.0 goftest_1.2-3 glue_1.6.2 nlme_3.1-162 promises_1.2.0.1
[61] grid_4.3.0 Rtsne_0.16 cluster_2.1.4 reshape2_1.4.4 generics_0.1.3 hdf5r_1.3.8
[67] gtable_0.3.3 spatstat.data_3.0-1 tidyr_1.3.0 data.table_1.14.8 sp_1.6-1 utf8_1.2.3
[73] spatstat.geom_3.2-1 RcppAnnoy_0.0.20 ggrepel_0.9.3 RANN_2.6.1 pillar_1.9.0 stringr_1.5.0
[79] later_1.3.1 splines_4.3.0 dplyr_1.1.2 lattice_0.21-8 renv_0.17.3 survival_3.5-5
[85] bit_4.0.5 deldir_1.0-9 tidyselect_1.2.0 miniUI_0.1.1.1 pbapply_1.7-0 gridExtra_2.3
[91] scattermore_1.1 shinydashboard_0.7.2 matrixStats_1.0.0 DT_0.28 stringi_1.7.12 lazyeval_0.2.2
[97] codetools_0.2-19 tibble_3.2.1 BiocManager_1.30.20 cli_3.6.1 uwot_0.1.14 xtable_1.8-4
[103] reticulate_1.28 munsell_0.5.0 Rcpp_1.0.10 globals_0.16.2 spatstat.random_3.1-5 png_0.1-8
[109] parallel_4.3.0 ellipsis_0.3.2 ggplot2_3.4.2 presto_1.0.0 listenv_0.9.0 viridisLite_0.4.2
[115] scales_1.2.1 ggridges_0.5.4 SeuratObject_4.1.3 leiden_0.4.3 purrr_1.0.1 crayon_1.5.2
[121] rlang_1.1.1 cowplot_1.1.1 shinyjs_2.1.0

[Gesmira]

Glad to hear it! As mentioned, we highly recommend clearing and restarting your R session before installing new versions of packages to ensure that it is a clean process in case previous versions are already loaded in.

[Gesmira]

Closing now as I believe the initial issue has been resolved. Please feel free to reopen if the error persists.

[mreza-ef]

I noticed that the reference must be a directory address containing the exact name of the "ext.Rds" file. I even encountered an error when the file was named "exr.rds."

[anglixue]

@mreza-ef Hi, I wonder where did you download the file "ext.Rds"? Thanks!

[ygq1983]

obj <- RunAzimuth(sce.qc_20, reference = "mousecortexref") Warning: Overwriting miscellanous data for model Warning: Adding a dimensional reduction (refUMAP) without the associated assay being present Warning: Adding a dimensional reduction (refUMAP) without the associated assay being present Error in curl::curl_fetch_memory(url, handle = handle) : Timeout was reached: [seurat.nygenome.org] Operation timed out after 60000 milliseconds with 0 bytes received