readfish stats

[alexomics]

          Dear Team,

I do have another issue. I tried to analyze the results using readfish stats with the --toml and --fastq-directory arguments. However, I see an error

(Channel 72 not found in Channel Map or', ' Barcode barcode48 not found in toml human_chr_selection.toml')

My toml file has the same content as the original human_chr_selection.toml file except for the file path to the reference mmi file. I suspect that I have to create a TOML table named barcodes and specify each of the barcodes within the toml file. Is this the way to go? Alternatively, could it be that the file path to the --fastq-directory is wrong? In this case, I specified the filepath to the run directory (e.g., /data/AFO_V3) or the file path to the fastq_pass directory (e.g., /data/AFO_V3/sample1/20240125_1411_MS00000_id_XXXXX/fastq_pass). I also tried to specify the file path to a specific barcode directory hosting fastq files (E.g., /data/AFO_V3/sample1/20240125_1411_MS00000_id_XXXXX/fastq_pass/barcode01). However, I still encountered the same error.

Originally posted by @ayoraind in https://github.com/LooseLab/readfish/issues/221#issuecomment-1918765115

[mattloose]

Hi,

You say you used the human_chr_selection.toml file - is this the one shown here:

https://github.com/LooseLab/readfish/blob/main/docs/_static/example_tomls/human_chr_selection.toml

This toml file is not necessarily compatible with barcoded runs for analysis with readfish stats - did you use this toml in the generation of the data? The data you are looking at are obviosuly barcoded and readfish stats will be assuming that (and inferring it from the reads).

If you did run adaptive sampling on a barcoded run with tthe human_chr_select.toml then we might need to tweak the readfish stats program to better handle this.

The adaptive sampling experiment should have worked regardless.

[ayoraind]

Yes, I used the human_chr_selection.toml file shown in the link you posted. I ran adaptive sampling using the human_chr_select.toml file.

The (stop-gap) solution was to

• copy lines 95-136 from the human_regions_barcoded.toml file (https://github.com/LooseLab/readfish/blob/main/docs/_static/example_tomls/human_regions_barcoded.toml) into the human_chr_select.toml file,
• change [barcodes.barcode01] to [barcodes.barcode48],
• run readfish validate to be sure it's valid,
• re-run the readfish stats command.

Seems to be working so far, although it is taking quite a while to align the FASTQ (currently at 2hr 33 minutes).

[mattloose]

OK - that's a plausible workaround. Let us know what the results look like - I'm not quite sure how it will behave.

[ayoraind]

Seems like it worked. I got the stats after 3hrs 54 minutes.

[bjaysheel]

Hi, What fastq-directory was used for stats, the fastq_pass directory or the root of output folder as you indicated in your first post?

[ayoraind]

Hi @bjaysheel,

I specified the file path to the run directory (e.g., /data/AFO_V3).

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