spl-IsoQuant with 10x Visium HD 3' ONT full-length spatial data ?

[Leilingshan]

Hello,

Thank you so much for developing such a fantastic tool!

I am currently preprocessing 10x Visium HD 3' Oxford Nanopore (ONT) full-length spatial transcriptomics data. According to the documentation for Spot-level UMI deduplication:

For spatial transcriptomics at higher resolution than the capture spots (e.g. Visium HD 2um barcodes mapping to 8um or 16um spots), use --barcode2barcode to deduplicate UMIs at the spot level:
isoquant.py --reference genome.fa --genedb genes.gtf --complete_genedb \
--fastq reads.fastq.gz --data_type nanopore \
--mode visium_hd --barcode_whitelist part1.txt part2.txt \
--barcode2barcode barcode2spot.tsv:0:1,2 \
-o visium_output
For Visium HD, generate the mapping file from per-part coordinate files:
python misc/prepare_visium_spot_ids.py part1_to_y.tsv part2_to_x.tsv -o barcode2spot.tsv

My question is, how can I obtain the part1_to_y.tsv and part2_to_x.tsv files?

I inspected the arguments in your misc/prepare_visium_spot_ids.py script, which states:

• part1: TSV file mapping barcode part1 to coordinate values (Y). Column 0: barcode sequence, columns 1..N: coordinate at each resolution.
• part2: TSV file mapping barcode part2 to coordinate values (X). Column 0: barcode sequence, columns 1..N: coordinate at each resolution.

However, when I looked into my local Space Ranger directory, the only HD-related file I could find is a binary/Protobuf file located at:
spaceranger-4.0.1/lib/python/cellranger/barcodes/visium_hd_v1.slide

Could you please guide me on how to extract or convert this visium_hd_v1.slide file into the part1_to_y.tsv and part2_to_x.tsv files required by your script? Or, how can I get the two files?

Additionally, do the part1.txt and part2.txt files required by the --barcode_whitelist parameter correspond directly to part1_to_y.tsv and part2_to_x.tsv respectively?

Lastly, if possible, could you kindly provide a detailed, step-by-step example of the full command-line workflow for running 10x Visium HD 3' ONT full-length spatial data?

Please excuse me if these questions seem a bit trivial, but I would highly appreciate any guidance or suggestions you could provide.

Thank you very much for your time and help!

Best regards,
Lingshan

[andrewprzh]

Dear @Leilingshan

Could you please write to me directly using my personal or isoquant email?
10x Visium HD barcodes are proprietary and are well hidden inside SpaceRanger. I am not sure it's nice to publish them here, at least not yet.

Best
Andrey

[andrewprzh]

To keep it public, the steps for Visium HD are described in our reproducibility repo: https://github.com/tilgnerlab/Spl-IsoFind_reproducibility/blob/main/SplIsoQuant.md

[Leilingshan]

Thank you very much for your reply. I will follow these steps to proceed with the analysis. @andrewprzh