# Matrices ## [Gene Expression File (GEF)](/saw-user-manual-v8.2/advanced/expression-matrix-format.md#gene-expression-file-gef) Gene expression file (GEF) is a data management and storage format designed to support multidimensional datasets and high computational efficiency. Stereo-seq analysis workflow generates bin GEF and cellbin GEF files. Bin GEF file format is a hierarchically structured data model that stores one or bin combined gene expression matrices in various bin sizes. Cellbin GEF file format stores expression information within each cell. Each GEF container organizes a collection of spatial gene expression matrices. It includes two primary data objects: Group and Dataset. A dataset is a multidimensional array of data elements. Group object is analogous to a file system directory that organizes datasets and other groups in hierarchies. ## [Gene Expression Matrix (GEM)](/saw-user-manual-v8.2/advanced/expression-matrix-format.md#gene-expression-matrix-gem) Gene expression matrix (GEM) stores gene spatial expression data. SAW generates multiple gene expression matrix files in the workflow, the basic format requires six columns with a header row that shows the column names. The six columns are gene ID, gene name, x coordinate, y coordinate, MID count and exon count. When it comes to cellbin GEM, the seventh column is for cell ID. The header of the expression matrix for the maximum area enclosing rectangle region has several annotation rows starting with "#" before the column rows. The header field names and field types are described in the table. ## File types The feature expression matrices generated from SAW pipelines mainly include two types, bin and cellbin GEF. They can be identified by the file extension:
File extensionDescription
.gef

The feature expression matrix file in HDF5 format for visualization. It contains the MID count for each gene of each spot. A spot is a binning unit that has a fixed-sized square shape in which the expression value in this square is accumulated. By default, a visualization .gef includes spot sizes of bin 1, 5, 10, 20, 50, 100, 150, 200.

.cellbin.gef

The cellbin feature expression matrix file in HDF5 format. It contains the spatial location and area of each cell, the MID count for each gene of each cell, and the cluster the cell belongs to. In .cellbin.gef, the cell is the smallest data unit.


Only available when the cell segmentation was done based on an microscopy image.

## Transcriptome Common output files of `SAW count` and `SAW realign` are listed:
FileDescription
<SN>.raw.gefFeature expression matrix includes the whole information over a complete chip region. It only has bin1 expression counts.
<SN>.gefFeature expression matrix. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
<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>_<stainType>_mask_edm_dis_<distance>.tif.
### Tissue statistics {% hint style="success" %} `.tissue.gef` is usually generated from a `.raw.gef` and a tissue segmentation image. {% endhint %} `tissuecut.stat` file can be found under `/STEREO_ANALYSIS_WORKFLOW_PROCESSING/EXPRESSION_MATRIX` and records statistics **under the detected tissue area**: | Metric | Description | | -------------------------------- | ----------------------------------------------------------------------------------------------------------------------------- | | Tissue area in square nanometers | The physical tissue area of the sample slice, in square nanometers. | | Contour area in pixel | The area of the tissue region on the tissue segmentation image, in pixels. | | Number of DNB under tissue | The number of detected DNBs with RNA capture under the tissue region. | | % of DNB under tissue | The proportion of detected DNBs with RNA capture under the tissue region relative to the total counts across the entire chip. | | Total gene type under tissue | The total number of annotated gene types under the tissue region. | | MID count under tissue | MID counts under the tissue region. | | % of MID under tissue | The proportion of MID counts under the tissue region relative to the total counts across the entire chip. | | Number of reads under tissue | The number of sequencing reads under the tissue region. | | % of reads under tissue | The proportion of sequencing reads under the tissue region relative to the total counts across the entire chip. | | Mean reads per spot (binN) | Mean reads of each binN spot under the tissue region. | | Median reads per spot (binN) | Median reads of each binN spot under the tissue region. | | Mean gene type per spot (binN) | Mean gene type of each binN spot under the tissue region. | | Median gene type per spot (binN) | Median gene type of each binN spot under the tissue region. | | Mean MID per spot (binN) | Mean MID count of each binN spot under the tissue region. | | Median MID per spot (binN) | Median MID count of each binN spot under the tissue region. | ## Microorganism If you perform `SAW count` on the Stereo-seq N FFPE and set `--microorganism-detect` to the analysis, its spatial expression matrices will be saved in `/outs/feature_expression/microorganism`. Output files are listed as:
FileDescription
<SN>.microorganism.raw.gefFeature expression matrix of microorganisms includes the whole information over a complete chip region. It only has bin1 expression counts.
<SN>.microorganism.gefFeature expression matrix of microorganisms. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
<SN>.host_microorganism.raw.gefFeature expression matrix of microorganisms and the host includes the whole information over a complete chip region. It only has bin1 expression counts.
<SN>.host_microorganism.gefFeature expression matrix of microorganisms and the host. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
<SN>.microorganism.<classification>.gem

Feature expression matrix of a specific classification of microbes.

Classifications of microorganisms include phylum, class, order, family, genus, and species.

### Microbe classification information After microbe classification via Kraken2, two files appear under `/STEREO_ANALYSIS_WORKFLOW/MICROOGANISM/ANALYSIS` are `seq_complete_info.txt` and `seq_complete_info_dedup.txt`. The difference between the two files is that the latter has undergone deduplication processing for microbe alignment. Each row represents a record of a read's alignment result, primarily including the read ID, spatial coordinate, MID count, taxonomic ID, scientific name, detailed biological classification, and read count. ```sh $ head ./STEREO_ANALYSIS_WORKFLOW_PROCESSING/MICROORGANISM/ANALYSIS/seq_complete_info.txt seq x y umi taxid Scientific_Name kindom phylum class order family genus species count V350264949L2C001R01701342667 11662 10937 ABB 77643 Mycobacterium_tuberculosis_complex k__Bacteria p__Actinobacteria c__Actinomycetia o__Corynebacteriales f__Mycobacteriaceae g__Mycobacterium 1 V350264949L2C001R01701347399 2742 13877 2B7 1783272 Terrabacteria_group k__Bacteria 1 V350264949L2C001R01900083155 18561 10644 C9E 5338 Agaricales k__Fungi p__Basidiomycota c__Agaricomycetes o__Agaricales 1 V350264949L2C001R01900086639 3861 14913 810 1760 Actinomycetia k__Bacteria p__Actinobacteria c__Actinomycetia 1 V350264949L2C001R01800770541 4264 17661 C20 1783272 Terrabacteria_group k__Bacteria 1 V350264949L2C001R01900181247 4396 16735 6EC 1762 Mycobacteriaceae k__Bacteria p__Actinobacteria c__Actinomycetia o__Corynebacteriales f__Mycobacteriaceae 1 V350264949L2C001R01900245227 18830 15878 D88 2 Bacteria k__Bacteria 1 V350264949L2C001R01900248762 18671 15840 77E 2 Bacteria k__Bacteria 1 V350264949L2C001R01900262154 15242 9034 4D0 1224 Proteobacteria k__Bacteria p__Proteobacteria 1 ``` ## Proteome If you perform `SAW count` on the Stereo-CITE T FF analysis, its spatial protein expression matrices will be saved in `/outs/feature_expression`. Output files are listed as:
FileDescription
<SN>.protein.raw.gefFeature expression matrix includes the whole information over a complete chip region. It only has bin1 expression counts.
<SN>.protein.gefFeature expression matrix. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
<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>_<stainType>_mask_edm_dis_<distance>.tif.
<SN>.protein.tissue.rmbg.gem.gzFeature expression matrix from automatic protein background removal. It shows bin1 expression counts.
### Tissue statistics {% hint style="success" %} `.protein.tissue.gef` is usually generated from a `.protein.raw.gef` and a tissue segmentation image. {% endhint %} `protein.tissuecut.stat` file can be found under `/STEREO_ANALYSIS_WORKFLOW_PROCESSING/EXPRESSION_MATRIX` and records statistics **under the detected tissue area**: | Metric | Description | | ----------------------------------- | ----------------------------------------------------------------------------------------------------------------------------- | | Tissue area in square nanometers | The physical tissue area of the sample slice, in square nanometers. | | Contour area in pixel | The area of the tissue region on the tissue segmentation image, in pixels. | | Number of DNB under tissue | The number of detected DNBs with ADT capture under the tissue region. | | % of DNB under tissue | The proportion of detected DNBs with ADT capture under the tissue region relative to the total counts across the entire chip. | | Total protein type under tissue | The total number of annotated protein types under the tissue region. | | MID count under tissue | MID counts under the tissue region. | | % of MID under tissue | The proportion of MID counts under the tissue region relative to the total counts across the entire chip. | | Number of reads under tissue | The number of sequencing reads under the tissue region. | | % of reads under tissue | The proportion of sequencing reads under the tissue region relative to the total counts across the entire chip. | | Mean reads per spot (binN) | Mean reads of each binN spot under the tissue region. | | Median reads per spot (binN) | Median reads of each binN spot under the tissue region. | | Mean protein type per spot (binN) | Mean protein type of each binN spot under the tissue region. | | Median protein type per spot (binN) | Median protein type of each binN spot under the tissue region. | | Mean MID per spot (binN) | Mean MID count of each binN spot under the tissue region. | | Median MID per spot (binN) | Median MID count of each binN spot under the tissue region. | aad --- # 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-v8.2/analysis/outputs/matrices.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.