Load Space Ranger 4.0 segmented outputs in Seurat

[Dena2021]

Hi Seurat team and community,

I’m working with the latest output from Space Ranger, which includes a segmented_outputs/ directory (e.g., cell_segmentations.geojson, filtered_feature_cell_matrix.h5, etc.), see https://www.10xgenomics.com/support/software/space-ranger/latest/analysis/outputs/segmented-outputs

Currently, it seems that Load10X_Spatial() does not support loading data from this folder directly.

Is there a recommended approach for importing the segmented outputs — including expression data and spatial coordinates — into Seurat for cell-level spatial analysis and visualization?

Thanks

[YoukaiFromAccounting]

Hello @Dena2021,

Thank you for your interest in the Seurat project; we are currently in the process of finalizing a new Load10X_Spatial function and subfunctions to include support for SpaceRanger 4.0 datasets. We estimate that we should have a working prototype branch to allow users to load in segmented_outputs in about a week or so.

Here's a sneak peek at the new Spaceranger objects visualized in Seurat:
Plotting with SpatialDimPlot (tissue image overlay with cell segmentations):

Zoomed in look at cell segmentations:

Gene expression image on spatial data:

[Dena2021]

Dear Satija lab team, Thank you for your amazing work! Looking forward to try this update.

…

Sent from my iPhone On 18 Jul 2025, at 17:49, YoukaiFromAccounting ***@***.***> wrote:  Caution: External Sender. Do not click on links or open attachments unless you recognize the sender. Hello @Dena2021<https://github.com/Dena2021>, Thank you for your interest in the Seurat project; we are currently in the process of finalizing a new Load10X_Spatial function and subfunctions to include support for SpaceRanger 4.0 datasets. We estimate that we should have a working prototype branch to allow users to load in segmented_outputs in about a week or so. Here's a sneak peek at the new Spaceranger objects visualized in Seurat: Plotting with SpatialDimPlot (tissue image overlay with cell segmentations): image.png (view on web)<https://github.com/user-attachments/assets/173fb50f-e15e-4feb-9e62-7ac4b8945679> Zoomed in look at cell segmentations: image.png (view on web)<https://github.com/user-attachments/assets/23c83937-8680-4ff4-b731-b41cf40f8be5> Gene expression image on spatial data: image.png (view on web)<https://github.com/user-attachments/assets/ee93337d-3547-49e6-8b2b-a556b2c701ab> — Reply to this email directly, view it on GitHub<#9991 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ASREESGQQY65HJ3YLDRUDQT3JECORAVCNFSM6AAAAACBM5TBLCVHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTGOBQGY4TQMA>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[kebbekny]

This looks incredible! Eagerly awaiting this update.

Best

[soumyanegi]

Thanks for working on this so quickly, Seurat team! Would love to test it out once available. Do let us know when. :)

[KarolinaBenkova]

Thank you for implementing this, I'm also looking forward. Just to clarify, are you using the CytAssist image as the image overlaid with the cell segmentations? I understand that the cell segmentations are done on the H&E images, however, seeing the overlay with the CytAssist image would be more helpful for me. Possibly, can you support both?

[YoukaiFromAccounting]

Hello @KarolinaBenkova, thank you very much for your message. Unfortunately, at the moment the cytassist_image.tiff and tissue_lowres_image.png are cropped and scaled completely differently, as well as varying between different experiments. This would make it very difficult to align with the coordinate space of the cell segmentations in a standardized way. Should Spaceranger 4.0 be updated to include an aligned cytassist_image.tiff, then we would be happy to include support for CytAssist image overlays.

Example of tissue_lowres_image.png:

Example of cytassist_image.tiff:

[KarolinaBenkova]

Hello @YoukaiFromAccounting, thank you for your answer and for the update. I have just messaged 10x regarding this issue. Interestingly, in the web summary Image Alignment tab, we can see the CytAssist to Microscope Image Alignment. I'm wondering whether the aligned CytAssist image existed when creating the summary, but is not part of the output files. I'm hoping for a positive answer from 10x :)

Edit: this has now been submitted to 10x as a feature request

[YoukaiFromAccounting]

Hello everyone,

A working branch for Seurat and Seurat object have just been released that contain support for reading in, loading, and visualizing Spaceranger 4.0 outputs and Visium HD 3' objects. Please refer to this pull request here, (#10028) which contains instructions on how to use existing Seurat functions with these new datasets.

As this is a branch in development, we anticipate that users may encounter a higher amount of warnings or errors. We appreciate everyone's patience as we continue development on more robust support of Visium HD 3' objects. Please report any issues you may encounter in Seurat's issue board.

We thank everyone for their continued interest in the Seurat project.