# Stereo-seq FF This tutorial will teach you how to run `SAW count` pipeline on the Stereo-seq chip derived from a fresh frozen (FF) mouse kidney. ## Prerequisites To run `SAW count` pipeline smoothly, you should: * Be acquainted with the Linux system. * Be familiar with running command line tools. * Ensure access to a system that meets the [minimum system requirements](/saw-user-manual-v8.0/download-center.md#system-requirements). {% hint style="info" %} **Adequate storage** and **sufficient permissions** should be paid more attention to, before running pipelines. {% endhint %} ## Overview of SAW count pipeline Stereo-seq sequencing data from FF tissues is analyzed with `SAW count`. The pipeline usually begins with: * a chip mask file (recording CIDs of the Stereo-seq chip), * FASTQ files (from Stereo-seq sequencing), * a reference (chosen by the organism information), * one or more microscope images (TIFF or image `.tar.gz` from **StereoMap**). {% hint style="info" %} The compressed image `.tar.gz` file, from StereoMap, saves the original microscope images and the QC information. {% endhint %}

Output results mainly include: * BAMs of alignment and annotation, * processed images, * gene expression matrices at different dimensions, * clustering and differential expression analysis, * an integrated `visualization.tar.gz` for **StereoMap**. ## Demo data Demo data of the mouse kidney from Stereo-seq Chip T is provided in this tutorial. Key features of demo - C02533C1: * Chip size: 1cm \* 1cm (S1) * Bin1: 500nm \* 500nm * Tissue section of 10μm thickness * H\&E-stained image acquired using Motic [The dataset page](https://en.stomics.tech/col1341/index) allows you to download the chip mask file, the raw sequencing files in FASTQ format, a TIFF image or an image `tar.gz`, a reference. For better organization, creating new folders for corresponding data is a wise choice. ```sh $ cd /saw # Create sub-folders of different datasets $ mkdir -p datasets/fastqs datasets/mask datasets/image datasets/reference ``` ## Command lines Set up `SAW count` analysis command in your working directory. ```sh saw count \ --id= \ --sn= \ --omics=transcriptomics \ --kit-version="Stereo-seq T FF V1.2" \ --sequencing-type="PE100_50+100" \ --chip-mask=/path/to/chip/mask \ --organism= \ --tissue= \ --fastqs=/path/to/fastq/folders \ --reference=/saw/datasets/reference \ --image-tar=/path/to/image/tar ``` Brief descriptions of the mentioned parameters in command lines:

Parameter	Description
`--id`	(Optional, default to None) A unique task id ([a-zA-Z0-9_-]+) which will be displayed as the output folder name and the title of HTML report. If the parameter is absent, `--sn` will play the same role.
`--sn <SN>`	(Required, default to None) SN (serial number) of the Stereo-seq chip.
`--omics <OMICS>`	(Required, default to "transcriptomics") Omics information.
`--kit-version <TEXT>`	(Required, default to None) The version of the product kit. More in count pipeline introduction.
`--sequencing-type <TEXT>`	(Required, default to None) Sequencing type of FASTQs which is recorded in the sequencing report.
`--chip-mask <MASK>`	(Required, default to None) Stereo-seq chip mask file.
`--organism <TEXT>`	(Optional, default to None) Organism type of sample, usually referring to species.
`--tissue <TEXT>`	(Optional, default to None) Physiological tissue of sample.
`--reference <PATH>`	(Optional, default to None) Path to the reference folder, containing SAW-compatible index files and GTF/GFF, built by `SAW makeRef`.
`--fastqs <PATH>`	(Required, default to None) Path(s) to folder(s), containing all needed FASTQs. If FASTQs are stored in multiple directories, use it as: `--fastqs=/path/to/directory1,/path/to/directory2,...`. Notice that all FASTQ files under these directories will be loaded for analysis.
`--image <TIFF>`	(Optional, default to None) TIFF image for QC (quality control), combined with expression matrix for analysis. Name rule for input TIFF : a. `<SN>_<stain_type>.tif` b. `<SN>_<stain_type>.tiff` c. `<SN>_<stain_type>.TIF` d. `<SN>_<stain_type>.TIFF` <stainType> includes: a. ssDNA b. DAPI c. HE (referring to H&E) d. <_IF_name1>_IF, <IF_name2>_IF, ...
`--image-tar <TAR>`	(Optional, default to None) The compressed image `.tar.gz` file from StereoMap has been through prepositive QC (quality control).

## Run SAW count Set up `SAW count` analysis command in your working directory. ```sh cd /saw/runs saw count \ --id=Demo_Mouse_Kidney \ --sn=C02533C1 \ --omics=transcriptomics \ --kit-version="Stereo-seq T FF V1.2" \ --sequencing-type="PE100_50+100" \ --chip-mask=/saw/datasets/mask/C02533C1.barcodeToPos.h5 \ --organism=mouse \ --tissue=kidney \ --fastqs=/saw/datasets/fastqs \ --reference=/saw/datasets/reference \ --image-tar=/saw/datasets/image/C02533C1_SC_20240327_201353_4.0.0.tar.gz ``` {% hint style="info" %} If you input a or certain images in TIFF, the prefixes of the file names should be: **\\_\\_\*.tif** When it comes to immunofluorescence staining (IF) image, that should be: **\\_\\_IF\_\*.tif** e.g.: * C04144D5\_ssDNA.tif * SS200000135TL\_D1\_DAPI.tif * C02533C1\_HE.tif (HE refers to H\&E-stained) * SS200000059\_NC\_DAPI.tif, SS200000059\_NC\_TESK2\_IF.tif, SS200000059\_NC\_APAK3\_IF.tif,... {% endhint %} ## Explore the output structure After pipeline analysis is completed, a new folder named `Demo_Mouse_Kidney` (which is provided by `--id`, or by `--sn` in the absence of `--id`) will appear in your working directory. All the metadata and outputs generated from `SAW count` are listed below: ``` Demo_Mouse_Kidney ├── pipeline-logs ├── STEREO_ANALYSIS_WORKFLOW_PROCESSING └── outs ├── analysis ├── bam ├── feature_expression ├── image ├── .report.tar.gz └── visualization.tar.gz ```

If you want to dig deeper into the results, * Jump to the [`report.html`](/saw-user-manual-v8.0/analysis/outputs/html-report.md) inside `.report.tar.gz`. * Explore the [`visualization.tar.gz`](https://stereotoolss-organization.gitbook.io/saw-user-manual-v8.0/tutorials/pages/w0RVrwTT5dQdztEbRw0t#visualization.tar.gz) in **StereoMap**. * Learn more about the individual files on the [Outputs](/saw-user-manual-v8.0/analysis/outputs.md) page. --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://stereotoolss-organization.gitbook.io/saw-user-manual-v8.0/tutorials/stereo-seq-ff.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.