Stereo-seq N FFPE

Stereo-seq kit information

SAW analyzes the sequencing data from the Stereo-seq chip for formalin-fixed paraffin-embedded (FFPE) samples. The workflow graph is shown below:

--kit-version for analysis is important to be known, according to the STOmics Stereo-seq Transcriptomics Set User Manual.

Stereo-seq Solution	Stereo-seq Kit	--kit-version
Stereo-seq Transcriptomics FFPE Solution	V1.0	"Stereo-seq N FFPE V1.0"

*N stands for Chip N

According to the relational reference table, confirm your --kit-version for the analysis.

Input files

SAW count pipeline requires input files as below:

• The mask file of the Stereo-seq Chip N (--mask)

• Stereo-seq sequencing FASTQ files (--fastqs)

--sequencing-type is related to FASTQs, for example, "PE75_25+59" indicates 75 bp of paired-end sequencing for the product kit, where the read length of read 1 is 25 and that of read 2 is 59. You can also find this information in the STOmics Stereo-seq Transcriptomics Set User Manual.

• The microscopic staining image in TIFF or .tar.gz format (from SteroMap)

  • --image for brightfield or fluorescent images in TIFF

  • --image-tar for brightfield or fluorescent image .tar.gz from StereoMap

• The reference, including FASTA genome and GTF/GFF annotation files for the species from which the sample was obtained (--reference)

  • reference index files should be built in advance, using SAW makeRef

  • if microorganism analysis is to be performed, additional reference index files should be built and databases should be downloaded in advance

  • annotation files will be checked for compliance with the standard format automatically, using SAW checkGTF

Run SAW count

For a list of available parameters, please refer to the command-line arguments, or simply type saw count --help. Before running the pipeline, ensure you have downloaded the chip mask file from our website, which is significant for analysis.

After preparation of input files, choose a suitable workflow according to the image QC result.

Automatic workflow

To generate spatial feature expression matrices for a Stereo-seq Chip from a formalin-fixed paraffin-embedded (FFPE) sample using automatic registration, tissue detection, cell segmentation and cell border expanding on a microscope image, run SAW count with the following arguments.

If the QC result of the input image is unsuccessful, SAW count will not call image-related algorithms for the processing, and will only perform tissue detection based on expression data.

Cell segmentation will not be called on an H&E-stained image of a FFPE tissue sample.

cd /saw/runs

saw count \
    --id=<task_id> \
    --sn=<SN> \
    --omics=transcriptomics \
    --kit-version="Stereo-seq N FFPE V1.0" \
    --sequencing-type="PE75_25+59" \
    --chip-mask=/path/to/chip/mask \
    --organism=<organism> \
    --tissue=<tissue> \
    --fastqs=/path/to/fastq/folders \
    --reference=/path/to/reference/folder \
    --image-tar=/path/to/image/tar

More in Stereo-seq FF tutorial.

Manual processing

After the first SAW count run, you can get the files from visualization.tar.gz for both presentation and manual processing in StereoMap. After a series of manual processes, an image .tar.gz will be passed back to SAW realign for the new results.

A QC-failed image should be performed registration between the image and the matrix in StereoMap. After that, SAW realign will restart the analysis, calling image-related algorithms based on the aligned image. But manually processed results (tissue and cell segmentation) from StereoMap will not be overwritten.

cd /saw/runs

saw realign \
    --id=<task_id2> \
    --sn=<SN> \
    --count-data=/path/to/previous/SAW/count/task/folder \
    --realigned-image-tar=/path/to/realigned/image/tar

More in Manual Processing tutorial.

Output files

All the metadata and outputs generated from SAW count are listed below:

Demo_Mouse_Brain
├── pipeline-logs
├── STEREO_ANALYSIS_WORKFLOW_PROCESSING
└── outs
    ├── analysis
    ├── bam
    ├── feature_expression
    ├── image
    ├── <SN>.report.tar.gz
    └── visualization.tar.gz

If you want to dig deeper into the results,

• Jump to the report.html inside <SN>.report.tar.gz.

• Explore the visualization.tar.gz in StereoMap.

• Learn more about the individual files on the Outputs page.