> For the complete documentation index, see [llms.txt](https://stereotoolss-organization.gitbook.io/saw-user-manual-v8.1/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://stereotoolss-organization.gitbook.io/saw-user-manual-v8.1/analysis/outputs/count-outputs.md). # count outputs ## Overview of output structure The `SAW count` pipeline runs in a directory named by `--id` (or by `--sn` in the absence of `--id`). Output files are classified into several folders, in the `outs/` directory. The exact output files generated from the analysis depend on: * the version of SAW used * which pipeline was used, `SAW count` or `SAW realign` * whether input the microscope image(s) * the specific parameters added to the analysis ## Spatial Gene Expression After performing `SAW count` on Stereo-seq T FF, Stereo-seq N FFPE, and Stereo-CITE T FF kits, the following files can be found under the `outs/` directory:
Directory/File NameDescription
bam/Files in BAM format.
annotated_bam/BAM file after alignment and annotation.
<SN>.*.bamIndexed BAM file containing position-sorted reads mapped to CIDs, aligned to the genome, and annotated with GTF/GFF.
<SN>.*.bam.csiIndex for <SN>.*.bam.
feature_expression/Feature expression matrices in HDF5 format at different dimensions.
<SN>.raw.gefFeature expression matrix includes the whole information over a complete chip region. It only has bin1 expression counts.
<SN>.tissue.gefFeature expression matrix under the tissue coverage region. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
<SN>.cellbin.gefCellbin feature expression matrix records the information of cells individually, including the centroid coordinate, boundary coordinates, expression of genes, and cell area.
<SN>.adjusted.cellbin.gefCellbin expression matrix with cell border expanding, based on <SN>_<stain_type>_mask_edm_dis_<distance>.tif.
<SN>.merge.barcodeReadsCount.txtA mapped CID list file with read counts for each CID, including three columns (x, y, count).
<SN>_raw_barcode_gene_exp.txtAn annotated list file with the information of coordinate, gene, MID, read counts, which is prepared to be a sampling file that performs sequence saturation.
analysis/Secondary analysis files.
<SN>.bin20_1.0.h5ad & <SN>.bin50_1.0.h5ad

An AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, based on <SN>.tissue.gef.

This output H5AD is named in the format of <SN>.<binN>_<leiden_res>.h5ad. In the file name, <SN> stands for the Stereo-seq chip serial number, <N> for bin size, and <leiden_res> for the resolution of Leiden clustering.

<SN>.bin20_1.0.marker_features.csv & <SN>.bin50_1.0.marker_features.csv Format-integrated differential expression analysis results, using <SN>.tissue.gef of bin20 and bin50.
<SN>.cellbin_1.0.h5adAn AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, using <SN>.cellbin.gef.
<SN>.cellbin_1.0.marker_features.csvFormat-integrated differential expression analysis results, using <SN>.cellbin.gef.
<SN>.cellbin_1.0.adjusted.h5adAn AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, using <SN>.adjusted.cellbin.gef.
<SN>.cellbin_1.0.adjusted.marker_features.csvFormat-integrated differential expression analysis results, using <SN>.adjusted.cellbin.gef.
## Spatial Protein Expression After performing `SAW count` on Strereo-CITE T FF kits, the following files can be found under the `outs/` directory:
Directory/File NameDescription
feature_expression/Feature expression matrices in HDF5 format at different dimensions.
<SN>.protein.raw.gefFeature expression matrix includes the whole information over a complete chip region. It only has bin1 expression counts.
<SN>.protein.tissue.gefFeature expression matrix under the tissue coverage region. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
<SN>.protein.cellbin.gefCellbin feature expression matrix records the information of cells individually, including the centroid coordinate, boundary coordinates, expression of genes, and cell area.
<SN>.protein.adjusted.cellbin.gefCellbin expression matrix with cell border expanding, based on <SN>_<stain_type>_mask_edm_dis_<distance>.tif.
<SN>.protein.tissue.rmbg.gem.gzFeature expression matrix from automatic protein background removal. It shows bin1 expression counts.
<SN>_cid_pid_mid_reads.tsvA list file with coordinate, PID, MID, and read counts, which is prepared to be a sampling file that performs sequence saturation for all proteins.
<SN>_valid_cid_reads.tsvA mapped CID list file from all ADT FASTQs, with read counts for each CID, including three columns (x, y, count).
analysis/Secondary analysis files.
<SN>.protein.bin20_0.1.h5ad & <SN>.protein.bin50_0.1.h5ad

An AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, based on <SN>.protein.tissue.gef.

This output H5AD is named in the format of <SN>.<binN>_<leiden_res>.h5ad. In the file name, <SN> stands for the Stereo-seq chip serial number, <N> for bin size, and <leiden_res> for the resolution of Leiden clustering.

<SN>.protein.cellbin_0.1.h5adAn AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, using <SN>.protein.cellbin.gef.
<SN>.protein.cellbin_0.1.adjusted.h5adAn AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, using <SN>.protein.adjusted.cellbin.gef.
## Image
Directory/File NameDescription
image/Images are generated from automatic or manual workflows.
<SN>_<stainType>_regist.tifThe panoramic image after the registration with <SN>.raw.gef matrix.
<SN>_<stainType>_tissue_cut.tifThe tissue segmentation image, based on the aligned panoramic image.
<SN>_<stainType>_mask.tifThe cell segmentation image, based on the aligned panoramic image.
<SN>_<stainType>_mask_edm_dis_<distance>.tifThe adjusted image, based on the cell segmentation image
## Report and Visualization
Directory/File NameDescription
<SN>.report.htmlAnalysis summary report of metrics and plots in HTML format.
visualization.tar.gzStereoMap visualization file to presentation and manual processing.
<SN>.stereoA manifest file in JSON format includes experiment and pipeline information, basic analysis statistics, and references to image and spatial matrix files in the SAW output visualization file folder.
## `visualization.tar.gz` The compressed visualization TAR file integrates all the output results needed by StereoMap for visualization. The contents of an unpacked one are listed: ``` visualization ├── C04042E3.adjusted.cellbin.gef ├── C04042E3.bin20_1.0.h5ad ├── C04042E3.bin50_1.0.h5ad ├── C04042E3.cellbin_1.0.adjusted.h5ad ├── C04042E3.rpi ├── C04042E3_SC_20240930_141410_4.1.0.tar.gz ├── C04042E3.stereo ├── C04042E3.tissue.gef └── HE_matrix_template.txt ``` The compressed visualization TAR file from Stereo-CITE analysis: ``` visualization ├── A02677B5.adjusted.cellbin.gef ├── A02677B5.bin20_1.0.h5ad ├── A02677B5.bin50_1.0.h5ad ├── A02677B5.cellbin_1.0.adjusted.h5ad ├── A02677B5.protein.adjusted.cellbin.gef ├── A02677B5.protein.bin20_0.1.h5ad ├── A02677B5.protein.bin50_0.1.h5ad ├── A02677B5.protein.cellbin_0.1.adjusted.h5ad ├── A02677B5.protein.tissue.gef ├── A02677B5.rpi ├── A02677B5_SC_20240930_094017_4.1.0.tar.gz ├── A02677B5.stereo ├── A02677B5.tissue.gef └── DAPI_matrix_template.txt ``` ### `.stereo` `.stereo` is a manifest file in JSON format that records * information about the task in SAW pipelines, * information of the tissue sample, * basic analysis statistics, * records of image files and expression data for **StereoMap** exploration. *\*More details about these files can be found in other parts of Outputs.*