Weird error loading while creating Visium HD giotto object

[alipirani88]

Hi,

Any idea what does this error mean?

visiumHD_object <- visiumHD$create_gobject(visiumHD_dir = "/Users/apirani/Documents/Data_projects/10X_Visium_HD_data/2024-07-23_H5_First_Batch/238687-HD01_275405-6-2/binned_outputs/square_002um", expr_data = "raw", gene_column_index = 2, png_name = "tissue_lowres_image.png")

Error:

python already initialized in this session
active environment : 'giotto_env'
python version : 3.10
a giotto python environment was found
Using python path:
"/Users/apirani/Library/r-miniconda-arm64/envs/giotto_env/bin/pythonw"
loading expression matrix ...
loading tissue positions file ...
creating visiumHD tissue position x expression data file ...
Selecting col "gene" as feat_ID column
Selecting cols "x" and "y" as x and y respectively
Setting feature info [rna]
loading expression matrix ...
loading tissue positions file ...
creating visiumHD tissue position x expression data file ...
subsetting visiumHD on array rows
subsetting visiumHD on array columns
subsetting visiumHD on expression matrix
Selecting col "gene" as feat_ID column
Selecting cols "x" and "y" as x and y respectively
Error in seq.default(x_start, e[["xmax"]] - (0.5 * ccd), by = ccd) :
wrong sign in 'by' argument

[alipirani88]

Also, I dont find any documentation for createGiottoVisiumHDObject function. Is this a typo in https://drieslab.github.io/Giotto_website/articles/VisiumHD_Human_Colorectal_Cancer.html

[RubD]

@iqraAmin have you seen this before?

[alipirani88]

I am also trying createGiottoVisiumObject but no luck. I get an error:

N_pros <- createGiottoVisiumObject(
+     visium_dir = "/Volumes/home/neuro_rsrch/apirani/10X_Visium_HD_data/2024-07-23_H5_First_Batch/238687-HD01_275405-6-2/binned_outputs/square_002um",
+     expr_data = "raw",
+     png_name = "tissue_lowres_image.png",
+     gene_column_index = 2,
+     instructions = instrs)
A structured visium directory will be used
Error in data.table::fread(tissue_positions_path) : 
  input= must be a single character string containing a file name, a system command containing at least one space, a URL starting 'http[s]://', 'ftp[s]://' or 'file://', or, the input data itself containing at least one \n or \r

[iqraAmin]

@iqraAmin have you seen this before?

I haven't come across an error like that before. The recent changes to the visiumHD functions haven't been merged into the suite branch yet; the latest version can be found in the suite_dev branch.

@alipirani88, could you provide me with the session info and a subset of your data so I can try to reproduce the error?

[alipirani88]

I am not sure how to subset the data with Giotto since I cannot generate a giotto object. I can subset it with Seurat if that would work or else if there is a better way to subset and share it - let me know.

FYI - I am able to manually create giotto object and generate an expression matrix using this code:

library(Giotto)
library(ComplexHeatmap)

setwd("/Users/apirani/Documents/Data_projects/10X_Visium_HD_data/2024-07-23_H5_First_Batch/238687-HD01_275405-6-2/binned_outputs")
save_dir <- "/Users/apirani/Documents/Data_projects/10X_Visium_HD_data_analysis/2024_10_15_Spatial_Giotto_analysis/2024_10_15_Primary_275405/plots"
dir.create(save_dir, recursive = TRUE)

data_path <- "/Users/apirani/Documents/Data_projects/10X_Visium_HD_data/2024-07-23_H5_First_Batch/238687-HD01_275405-6-2/binned_outputs"

expression_path <- file.path(data_path, 'square_002um/filtered_feature_bc_matrix')
expr_results <- get10Xmatrix(path_to_data = expression_path, 
                             gene_column_index = 2)


tissue_positions_path <- file.path(data_path, 'square_002um/spatial/tissue_positions.parquet')
tissue_positions <- data.table::as.data.table(arrow::read_parquet(tissue_positions_path))

# convert expression matrix to minimal data.frame or data.table object
matrix_tile_dt <- data.table::as.data.table(Matrix::summary(expr_results))
genes   <- expr_results@Dimnames[[1]]
samples <- expr_results@Dimnames[[2]]
matrix_tile_dt[, gene := genes[i]]
matrix_tile_dt[, pixel := samples[j]]


expr_pos_data <- data.table::merge.data.table(matrix_tile_dt, 
                                             tissue_positions, 
                                             by.x = 'pixel', 
                                             by.y = 'barcode')

expr_pos_data <- expr_pos_data[,.(pixel, pxl_row_in_fullres, pxl_col_in_fullres, gene, x)]
colnames(expr_pos_data) = c('pixel', 'x', 'y', 'gene', 'count')

giotto_points = createGiottoPoints(x = expr_pos_data[,.(x, y, gene, pixel, count)])

hexbin400 <- tessellate(extent = ext(giotto_points), 
                        shape = 'hexagon', 
                        shape_size = 400, 
                        name = 'hex400') 
plot(hexbin400)

instrs = createGiottoInstructions(
  save_dir = save_dir,
  save_plot = TRUE,
  show_plot = FALSE,
  return_plot = FALSE
)

# gpoints provides spatial gene expression information
# gpolygons provides spatial unit information (here = hexagon tiles)
visiumHD = createGiottoObjectSubcellular(gpoints = list('rna' = giotto_points),
                                         gpolygons = list('hex400' = hexbin400),
                                         instructions = instrs)

# create spatial centroids for each spatial unit (hexagon)
visiumHD = addSpatialCentroidLocations(gobject = visiumHD,
                                       poly_info = 'hex400')

Is there a way to create Giotto visium object using the above code instead of createGiottoObjectSubcellular?

Downstream I would like to merge two objects from treated vs non-treated groups to perform integration analysis.

[iqraAmin]

@alipirani88, could you share which Giotto version you’re using? The createGiottoVisiumHDObject() function is available in the newer Giotto version (4.1.3) on the suite_dev branch.

[alipirani88]

Sure - Here is my session information

R version 4.4.1 (2024-06-14)
Platform: aarch64-apple-darwin20
Running under: macOS Sonoma 14.7

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/Chicago
tzcode source: internal

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

other attached packages:
[1] ComplexHeatmap_2.20.0 Giotto_4.1.0          GiottoClass_0.3.5    

loaded via a namespace (and not attached):
  [1] colorRamp2_0.1.0      deldir_2.0-4          rlang_1.1.4           magrittr_2.0.3        clue_0.3-65           GetoptLong_1.0.5      GiottoUtils_0.1.12    matrixStats_1.4.1    
  [9] compiler_4.4.1        png_0.1-8             systemfonts_1.1.0     vctrs_0.6.5           reshape2_1.4.4        stringr_1.5.1         pkgconfig_2.0.3       shape_1.4.6.1        
 [17] crayon_1.5.3          fastmap_1.2.0         XVector_0.44.0        backports_1.5.0       magick_2.8.5          labeling_0.4.3        utf8_1.2.4            rmarkdown_2.28       
 [25] ragg_1.3.3            purrr_1.0.2           bit_4.5.0             xfun_0.48             beachmat_2.20.0       zlibbioc_1.50.0       cachem_1.1.0          jsonlite_1.8.9       
 [33] DelayedArray_0.30.1   BiocParallel_1.38.0   terra_1.7-78          irlba_2.3.5.1         parallel_4.4.1        cluster_2.1.6         R6_2.5.1              bslib_0.8.0          
 [41] stringi_1.8.4         RColorBrewer_1.1-3    reticulate_1.39.0     parallelly_1.38.0     jquerylib_0.1.4       scattermore_1.2       Rcpp_1.0.13           assertthat_0.2.1     
 [49] iterators_1.0.14      knitr_1.48            future.apply_1.11.2   R.utils_2.12.3        FNN_1.1.4.1           IRanges_2.38.1        Matrix_1.7-0          igraph_2.0.3         
 [57] tidyselect_1.2.1      abind_1.4-8           rstudioapi_0.16.0     yaml_2.3.10           doParallel_1.0.17     codetools_0.2-20      listenv_0.9.1         lattice_0.22-6       
 [65] tibble_3.2.1          plyr_1.8.9            withr_3.0.1           evaluate_1.0.0        future_1.34.0         circlize_0.4.16       pillar_1.9.0          MatrixGenerics_1.16.0
 [73] checkmate_2.3.2       foreach_1.5.2         stats4_4.4.1          plotly_4.10.4         generics_0.1.3        dbscan_1.2-0          sp_2.1-4              S4Vectors_0.42.1     
 [81] ggplot2_3.5.1         munsell_0.5.1         scales_1.3.0          globals_0.16.3        gtools_3.9.5          glue_1.8.0            lazyeval_0.2.2        tools_4.4.1          
 [89] GiottoVisuals_0.2.4   data.table_1.16.0     ScaledMatrix_1.12.0   cowplot_1.1.3         tidyr_1.3.1           colorspace_2.1-1      BiocSingular_1.20.0   rsvd_1.0.5           
 [97] cli_3.6.3             textshaping_0.4.0     fansi_1.0.6           S4Arrays_1.4.1        viridisLite_0.4.2     arrow_17.0.0.1        dplyr_1.1.4           uwot_0.2.2           
[105] gtable_0.3.5          R.methodsS3_1.8.2     sass_0.4.9            digest_0.6.37         BiocGenerics_0.50.0   SparseArray_1.4.8     ggrepel_0.9.6         farver_2.1.2         
[113] rjson_0.2.23          htmlwidgets_1.6.4     htmltools_0.5.8.1     R.oo_1.26.0           lifecycle_1.0.4       httr_1.4.7            GlobalOptions_0.1.2   bit64_4.5.2      

I tried installing the suite_dev branch (devtools::install_github("drieslab/Giotto@suite_dev")) but I get this error:

Installing package into ‘/Users/apirani/Library/R/arm64/4.4/library’
(as ‘lib’ is unspecified)
ERROR: dependencies ‘GiottoClass’, ‘GiottoUtils’, ‘GiottoVisuals’ are not available for package ‘Giotto’
* removing ‘/Users/apirani/Library/R/arm64/4.4/library/Giotto’
Warning messages:
1: packages ‘GiottoVisuals’, ‘GiottoUtils’, ‘GiottoClass’ are not available for this version of R

Versions of these packages for your version of R might be available elsewhere,
see the ideas at
https://cran.r-project.org/doc/manuals/r-patched/R-admin.html#Installing-packages 
2: In i.p(...) :
  installation of package ‘/var/folders/3w/d12xrph92qzffw5mw2y34kk80000gq/T//RtmpjSh84N/file867f2aa18e6b/Giotto_4.1.3.tar.gz’ had non-zero exit status

[iqraAmin]

@alipirani88, could you try installing GiottoVisuals@dev, GiottoUtils@dev, and GiottoClass@dev first? Once those are installed, please proceed with the Giotto@suite_dev installation. Hopefully, this will resolve the issue.