Load10X_Spatial function error

[MJoseMo]

Hi,

We are trying to reproduce the results from the paper: Spatially resolved transcriptomics reveals the architecture of the tumor-microenvironment interface, We have downloaded the data from GEO. The data provided is: GSM4838131_Visium_Sample_A_filtered_feature_bc_matrix.h5, GSM4838131_Visium_A_image.tif.gz, GSM4838131_Visium_Sample_A_tissue_positions_list.csv.gz. (spatial folder)

We couldn't read the data in R with load10X_Spatial function from Seurat package.

[anashen]

Hello @MJoseMo,

To help us diagnose the issue, can you please reply and share:

• How the data from GEO is set up in your directory
• How you called the Load10X_Spatial function
• Any errors you may have received
• The result of running sessionInfo()

Thanks!

[MJoseMo]

Dear @anashen

The code is the next one:

A1_new <- Load10X_Spatial(
data.dir = "C:/Users/MM.5076373/Downloads/GSE159709_RAW/A1",
image = "GSM4838131_Visium_A_image.tif"
)

Error en FUN(X[[i]],...):File not found

[anashen]

Thank you @MJoseMo.

You will need thefiltered_feature_bc_matrix.h5 file in the directory passed to Load10X_Spatial. See the function documentation: https://satijalab.org/seurat/reference/load10x_spatial.

So, say you have a folder named "A1", with the path to A1 being "C:/Users/MM.5076373/Downloads/GSE159709_RAW/A1" from your code above. The folder should contain "spatial/" and "filtered_feature_bc_matrix.h5". Then the following call to Load10X_Spatial should be successful:

A1_new <- Load10X_Spatial(
data.dir = "C:/Users/MM.5076373/Downloads/GSE159709_RAW/A1"
)

Hope this helps - please let me know if you have any follow ups.

[MJoseMo]

@anashen , This is my working directory, I have different folders with the same data, I don't know how it need to be structured

[anashen]

@MJoseMo Thanks for following up - the default settings for Load10X_Spatial assume that the h5 file is named "filtered_feature_bc_matrix.h5" (again, noted in the documentation).

So you will need to specify it since your file is named differently:

A1_new <- Load10X_Spatial(data.dir,
                        filename = "GSM4838131_Visium_Sample_A_filtered_feature_bc_matrix.h5")

[MJoseMo]

thanks @anashen , for imagen, I would like to ask if it need to be in png format, or could be used other format as .tif and it need to be tissue_lowres_image or other name could be used.

Error en value[[3L]](cond): 
  Neither primary nor fallback image could be read:
C:/Users/MM.5076373/Downloads/GSE159709_RAW/A1/spatial/tissue_lowres_image.png
C:/Users/MM.5076373/Downloads/spatial/tissue_lowres_image.png

[anashen]

@MJoseMo the image needs to be a PNG, you can pass either "tissue_lowres_image.png" (default) or "tissue_hires_image.png".

[MJoseMo]

the tissue_lowres_image.png is already in the spatial folder.