CRUK-CI Genomics Help

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submission:10x_visium [2025/02/13 12:10] – skelto01submission:10x_visium [2025/02/14 14:53] (current) – [Overview of the Visium & Visium HD workflows] Ania Piskorz
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 ====== 10x Genomics Visium Submission======= ====== 10x Genomics Visium Submission=======
- 10X Visium samples are processed across both the Histopathology and Genomics cores, so prior to submission, please do contact [[singlecell@cruk.cam.ac.uk|single cell team]]  [[histopathology@cruk.cam.ac.uk|histology]]+ 10X Visium samples are processed across both the Histopathology and Genomics cores, so prior to submission, please do contact [[singlecell@cruk.cam.ac.uk|Single Cell Team]]  [[histopathology@cruk.cam.ac.uk|Histology]] 
 + 
 +We would recommend arranging to discuss your experimental aims with both teams by discussing this at a experimental design meeting (EDM): 
 +[[experimentaldesign@cruk.cam.ac.uk|Request EDM Here]] 
 +=====Overview of the Visium & Visium HD workflows===== 
 +Visium provides a whole transcriptome snapshot of gene expression across a tissue section, allowing you to identify spatial patterns of gene expression within different cell types and tissue regions. With Visium HD a significantly higher resolution is achievable essentially providing "single-cell scale" analysis by capturing gene expression data within very small, densely packed regions of a tissue section when compared to the larger spatial bins of "standard" (SD) Visium (1-10cells).  
 + 
 +{{:submission:vishd1.png?800|}}
  
-We would also recommend arranging to discuss your experimental aims with both teams by discussing this at a experimental design meeting (EDM): 
-[[experimentaldesign@cruk.cam.ac.uk|request EDM here]] 
-=====Overview of the Visium workflows===== 
 {{:visium_workflow2j.png?920|}} {{:visium_workflow2j.png?920|}}
  
 =====Visium options available===== =====Visium options available=====
 ^Workflow ^Kit Used ^Slide used for submission ^Method of mRNA capture ^Spot-based array | ^Workflow ^Kit Used ^Slide used for submission ^Method of mRNA capture ^Spot-based array |
-|**Visium HD** (FFPE) \\ |Visium HD slide & Cassette, Visium HD reagent kit, Visium Human or mouse probe kit|6.5 x6.5 mm |Whole transcriptome probe capture (Human & Mouse probe kits available)|Spot size: 2um| +|**Visium HD** (FFPE) \\ |Visium HD slide & Cassette, Visium HD reagent kit, Visium Human or mouse probe kit|6.5 x6.5mm section fixed on a regular superfrost slide|Whole transcriptome probe capture \\ (Human & Mouse probe kits available)|lawn of 2um, recommended bin size for analysis 8uM, single cell resolution| 
-|**Visium HD** (fresh frozen)\\ |Visium HD Slide & Cassette, Visium HD reagent kit, Visium Human or Mouse Probe Kit|6.5 x 6.5mm|Whole Transcriptome probe capture (Human & Mouse probe kits avalible)|Spot size: 2um| +|**Visium HD**\\ (fresh frozen)|Visium HD Slide & Cassette, Visium HD reagent kit, Visium Human or Mouse Probe Kit|6.5 x6.5mm section fixed on a regular superfrost slide|Whole Transcriptome probe capture \\ (Human & Mouse probe kits available)|lawn of 2um, recommended bin size for analysis 8uM, single cell resolution| 
-|**Visium CytAssist**\\ (FFPE)|Visium Slide & cassette, FFPE Reagent Kit, Probe Kit|FFPE tissue section of either 11 x 11 mm or 6.5 x 6.5mm in area, fixed onto regular superfrost slide|Whole Transcriptome probe capture (Human & Mouse probe kits available)|Spot size 55um\\ 11 x 11mm 14000 spots\\ 6.5mm x 6.5mm 5000 spots| +|**Visium CytAssist**\\ (FFPE)|Visium Slide & cassette, FFPE Reagent Kit, Probe Kit|FFPE tissue section of either 11 x 11 mm or 6.5 x 6.5mm in area, fixed onto regular superfrost slide|Whole Transcriptome probe capture\\ (Human & Mouse probe kits available)|Spot size 55um\\ 11 x 11mm 14000 spots\\ 6.5mm x 6.5mm 5000 spots, average 1-10 cells resolution| 
-|**Visium CytAssist**\\ (Fresh Frozen)|Visium Slide & cassette, FFPE Reagent Kit, Probe Kit|Fresh tissue section of either 11 x 11mm or 6.5 x 6.5 mm in area, fixed onto regular superfrost slide|Whole Transcriptome probe capture (Human & Mouse probe kits available)|Spot size 55um\\ 11 x 11mm 14000 spots\\ 6.5mm x 6.5mm 5000 spots| +|**Visium CytAssist**\\ (Fresh Frozen)|Visium Slide & cassette, FFPE Reagent Kit, Probe Kit|Fresh tissue section of either 11 x 11mm or 6.5 x 6.5 mm in area, fixed onto regular superfrost slide|Whole Transcriptome probe capture \\ (Human & Mouse probe kits available)|Spot size 55um\\ 11 x 11mm 14000 spots\\ 6.5mm x 6.5mm 5000 spots, average 1-10 cells resolution| 
-|**Visium Spatial Gene Expression Manual**\\ (FFPE)|Visium Spatial Gene Expression Slide Kit, Visium FFPE Reagent Kit, Probe Kit|4 capture areas available for tissue sections 6.5 x 6.5mm in area, fixed onto a spatial gene expression slide (provided by 10X) |Whole Transcriptome probe capture (Human & Mouse probe kits available)|Spot size: each capture area has ~5000 spots| +|**Visium Spatial Gene Expression Manual**\\ (FFPE)|Visium Spatial Gene Expression Slide Kit, Visium FFPE Reagent Kit, Probe Kit|4 capture areas available for tissue sections 6.5 x 6.5mm in area, fixed onto a spatial gene expression slide\\ (**provided by 10X**) |Whole Transcriptome probe capture \\ (Human & Mouse probe kits available)|Spot size: each capture area has ~5000 spots, average 1-10 cells resolution| 
-|**Visium Spatial Gene Expression Manual**\\ (Fresh Frozen)|Visium Spatial Gene Expression Slide & Kit, Probe Kit|4 capture areas available for tissue sections 6.5 x 6.5mm in area, fixed onto a spatial gene expression slide (provided by 10X) |Poly(A) tail capture |Spot size: each capture area has ~5000 spots| +|**Visium Spatial Gene Expression Manual**\\ (Fresh Frozen)|Visium Spatial Gene Expression Slide & Kit, Probe Kit|4 capture areas available for tissue sections 6.5 x 6.5mm in area, fixed onto a spatial gene expression slide\\ (**provided by 10X**) |Poly(A) tail capture |Spot size: each capture area has ~5000 spots, average 1-10 cells resolution| 
-|**Visium CytAssist Spatial Gene & Protein Expression**|Visium Slide & Cassette, FFPE Reagent Kit, Probe Kit, Immune Cell Profiling Panel, Add on antibodies (if applicable), Visium Protein Core Reagents (CITESeq etc)|FFPE tissue section of either 6.5 x 6.5mm in area, fixed onto regular superfrost slide|Whole Transciptome probe capture (Human & Mouse probe kits available) |Spot size: 11 x 11mm 14000 spots\\ 6.5mm x 6.5mm 5000 spots|+|**Visium CytAssist Spatial Gene & Protein Expression**|Visium Slide & Cassette, FFPE Reagent Kit, Probe Kit, Immune Cell Profiling Panel, Add on antibodies (if applicable), Visium Protein Core Reagents (CITE-Seq)|FFPE tissue section of either 6.5 x 6.5mm in area, fixed onto regular superfrost slide|Whole Transciptome probe capture \\ (Human & Mouse probe kits available) |Spot size: 11 x 11mm 14000 spots\\ 6.5mm x 6.5mm 5000 spots, average 1-10 cells resolution|
  
-Make sure you provide additional H&E section or electronic version of H&E image with marked area of interest (ROI) according to the selected workflow (11 x 11mm or 6.5 x 6.5mm) to guide 10X Visium probe hybridisation and library construction.+Make sure you provide additional H&E section or electronic version of H&E image with marked area of interest (ROI) according to the selected workflow (11 x 11mm or 6.5 x 6.5mm) to guide placement of the gasket and the alignment of the superfrost slide against the Visium slide capture array.
  
 =====How to Complete the 10x Visium Submission Form===== =====How to Complete the 10x Visium Submission Form=====
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   * **Name:** Name of your sample.    * **Name:** Name of your sample. 
-  * **RGT Number:** This ID is related to human tissue and is generated when sample is processed by Tissue Bank or being submitted to CRUK CI (using Achiver Medical). Format of RGT00xxxxxx.+  * **RGT Number:** This ID is related to human tissue and is generated when sample is processed by Tissue Bank or being submitted to CRUK CI (using Achiever Medical). Format of RGT00xxxxxx.
   * **RGT Aliquot Number:** This provided by histopathology or Tissue Bank (if they generate aliquots of original tissue). Format of three-digit code after the RGT number: RGT00xxxxxx-001 (leave empty if you do not have that information).    * **RGT Aliquot Number:** This provided by histopathology or Tissue Bank (if they generate aliquots of original tissue). Format of three-digit code after the RGT number: RGT00xxxxxx-001 (leave empty if you do not have that information). 
   * **Slide ID:** Any additional slide ID provided eg. CRU number or what is written on the slide.    * **Slide ID:** Any additional slide ID provided eg. CRU number or what is written on the slide. 
   *  **Capture Area (%):** If it is known what percentage of the 6.5 x 6.5mm or 11 x 11mm capture area your tissue covers, please write this here (leave empty if you do not have that information).   *  **Capture Area (%):** If it is known what percentage of the 6.5 x 6.5mm or 11 x 11mm capture area your tissue covers, please write this here (leave empty if you do not have that information).
-  * **DV200:** Please provide this if you have done an RNA extraction and Tapestation QC on a tissue section from the same block, to give us an idea of the RNA quality of your tissue sample (leave empty if you do not have that information).+  * **DV200:** Please provide this if you have done an RNA extraction and TapeStation QC on a tissue section from the same block, to give us an idea of the RNA quality of your tissue sample (leave empty if you do not have that information).
   * **Species:** only complete this if it is different from the species provided in the sample set information.   * **Species:** only complete this if it is different from the species provided in the sample set information.
-  * **Reads per spot:** How many sequencing reads do you want per spot of the visium slide? – this indicates the depth of sequencing you would like. +  * **Reads per spot:** How many sequencing reads do you want per spot of the Visium slide? – this indicates the depth of sequencing you would like. 
  
-For Visium HD:  +For Visium HD, 10x Genomics recommend 275M reads per capture region. 
-For standard Visium with Cytassist or manual: 10x suggest 25,000 reads per spot for FFPE tissue, and 50,000 reads per spot for fresh frozen on their guidelines but it is up to you! How many reads per spot you want does influence what flow cell you will need to use for sequencing, so if you would like some guidance, please do contact the single cell team who will be able to advise you. +For standard Visium with CytAssist or manual, 10x Genomics suggest 25,000 reads per spot for FFPE tissue, and 50,000 reads per spot for fresh frozen on their guidelines but it is up to you! How many reads per spot you want does influence what flow cell you will need to use for sequencing, so if you would like some guidance, please do contact the Single Cell team who will be able to advise you. 
  
 ===== 10X Genomics sequencing configuration recommendations===== ===== 10X Genomics sequencing configuration recommendations=====