# realign outputs ## Overview of output structure The `SAW realign` 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 realign` on Stereo-seq T FF and Stereo-seq N FFPE kits, the following files can be found under the `outs/` directory: | Directory/File Name | Description | | ---------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | **`bam/`** | Files in BAM format. | | `annotated_bam/` | BAM file after alignment and annotation. | | `.*.bam` | Indexed BAM file containing position-sorted reads mapped to CIDs, aligned to the genome, and annotated with GTF/GFF. | | `.*.bam.csi` | Index for `.*.bam`. | | **`image/`** | Images are generated from automatic or manual workflows. | | `__regist.tif` | The panoramic image aligned with `raw.gef` matrix. | | `__tissue_cut.tif` | The tissue segmentation image, based on the aligned panoramic image. | | `__mask.tif` | The cell segmentation image, based on the aligned panoramic image. | | `__mask_edm_dis_.tif` | The adjusted image, based on the cell segmentation image | | **`feature_expression/`** | Feature expression matrices in HDF5 format at different dimensions. | | `.raw.gef` | Feature expression matrix includes the whole information over a complete chip region. It only has bin1 expression counts. | | `.tissue.gef` | Feature expression matrix under the tissue coverage region. It is also a visualization GEF which includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200. | | `.cellbin.gef` | Cellbin feature expression matrix records the information of cells individually, including the centroid coordinate, boundary coordinates, expression of genes, and cell area. | | `.adjusted.cellbin.gef` | Cellbin expression matrix with cell border expanding, based on `__mask_edm_dis_.tif`. | | `.merge.barcodeReadsCount.txt` | A mapped CID list file with read counts for each CID, including three columns (x, y, count). | | `_raw_barcode_gene_exp.txt` | An 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. | | `.bin200_1.0.h5ad` |

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

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

| | `bin200_marker_features.csv` | Format-integrated differential expression analysis results, using `.tissue.gef` of bin200. | | `.cellbin_1.0.h5ad` | An AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, using `.cellbin.gef`. | | `cellbin_marker_features.csv` | Format-integrated differential expression analysis results, using `.cellbin.gef`. | | `.cellbin_1.0.adjusted.h5ad` | An AnnData H5AD records preprocessing, filtering, normalization, dimensionality reduction, clustering and differential expression analysis, using `.adjusted.cellbin.gef`. | | `cellbin_adjusted_marker_features.csv` | Format-integrated differential expression analysis results, using `.adjusted.cellbin.gef`. | | **`.report.tar.gz`** | Analysis summary report of metrics and plots in HTML format. | | `report.html` | HTML file, involved in `.report.tar.gz`. | | **`visualization.tar.gz`** | StereoMap visualization file to presentation and manual processing. | | `.stereo` | A 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. | {% hint style="info" %} Expression-related data is from the last `SAW count` output directory, through `--count-data` parameter. {% endhint %} ## `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 ├── C04144D5.adjusted.cellbin.gef ├── C04144D5.bin200_1.0.h5ad ├── C04144D5.cellbin_1.0.adjusted.h5ad ├── C04144D5.rpi ├── ssDNA_matrix_template.txt ├── C04144D5_SC_20240509_174202_4.0.0.tar.gz ├── C04144D5.stereo └── C04144D5.tissue.gef ``` ### `.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.*