Stereo-CITE T FF

Stereo-CITE kit information

STOmics Stereo-CITE Proteo-Transcriptomics Set is intended for simultaneous detection of the whole transcriptome and 100+ plex protein on the same tissue section. SAW analyzes the sequencing data from the Stereo-seq chip for fresh frozen (FF) samples. The workflow graph shown below:

--kit-version for analysis is important to be known, according to the Stereo-CITE Proteo-Transcriptomics User Manual.

Stereo-seq Solution	Stereo-CITE Kit	libraries	--kit-version	--sequencing-type
Stereo-CITE Proteo-Transcriptomics Solution	V1.0	pooling	"Stereo-CITE T FF V1.0"	"PE100_50+100"
Stereo-CITE Proteo-Transcriptomics Solution	V1.0	sequence Transcriptome and ADT libraries separately	"Stereo-CITE T FF V1.0"	"PE100_50+100","PE100_50+36"
Stereo-CITE Proteo-Transcriptomics Solution	V1.1	pooling	"Stereo-CITE T FF V1.1"	"PE75_50+100"
Stereo-CITE Proteo-Transcriptomics Solution	V1.1	sequence Transcriptome and ADT libraries separately	"Stereo-CITE T FF V1.1"	"PE75_50+100", "PE75_50+36"

*T stands for Chip T

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 T (--mask)

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

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

--sequencing-type is related to FASTQs, for example, "PE100_50+100" indicates 100 bp of paired-end sequencing for the product kit, where the read length of read 1 is 50 and that of read 2 is 100. You can find this information in the sequencing report.

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

  • --image for fluorescent images in TIFF

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

• The reference library in a CSV file (--ref-libraries)

  • The protein panel, including the antibody reference. The protein panel file can be found in Protein panels

  • Transcriptome reference folder, including the FASTA genome and GTF/GFF annotation files for the sample species

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

    • 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-CITE Chip from a fresh frozen (FF) 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.

cd /saw/runs

saw count \
    --id=<task_id> \
    --sn=<SN> \
    --omics=transcriptomics,proteomics \
    --kit-version="Stereo-CITE T FF V1.1" \
    --sequencing-type="PE75_50+100" \
    --chip-mask=/path/to/chip/mask \
    --organism=<organism> \
    --tissue=<tissue> \
    --fastqs=/saw/datasets/STOmics-RNA-fastqs \
    --adt-fastqs=/saw/datasets/STOmics-ADT-fastqs \
    --ref-libraries=/saw/datasets/reference/ref_libraries.csv \
    --image-tar=/path/to/image/tar

More in Stereo-CITE 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_Kidney
├── pipeline-logs
├── STEREO_ANALYSIS_WORKFLOW_PROCESSING
└── outs
    ├── analysis
    ├── bam
    ├── feature_expression
    ├── image
    ├── <SN>.report.html
    └── 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.