A problem in using REDI3 analyze -e

[1127yangyuping]

Thank you for developing REDItools3, which is a very valuable tool for RNA editing analysis. I have a couple of questions that I hope you can help me with.

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Question 1 - Regarding the -e Parameter and Multiple Substitution Sites:

"I am currently using the analyze module with the -e parameter in my command:

python -m reditools analyze \
    "$bam" \
    -r "$REF" \
    -o "$outfile" \
    -t "$THREADS" \
    -l 10 \
    -me 3 \
    -bq 30 \
    -q 20 \
    -e \
    -v all \
    -sf splicing.bed \
    -ss 4

When I use the -e flag, it does not appear to effectively exclude or filter out sites with multiple substitutions (multi-substituted sites). Could you please clarify:

• What is the exact function of the -e parameter?
• Is it supposed to exclude/remove multi-substituted sites from the output?
• If so, why might it not be working as expected in my analysis?
• Are there any specific conditions or additional parameters needed for it to function properly?"

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Question 2 - Regarding the -sf Parameter BED File Format:

"I also have a question about the BED file format required for the -sf (splicing sites file) parameter. Could you please specify the exact format specifications for this input file? Specifically:

• What are the required columns in the BED file (3-column, 6-column, or other)?
• Are there any additional columns needed beyond the standard BED format?
• What coordinate system should be used (0-based or 1-based)?
• Are there any specific requirements or conventions for the feature names or annotations in this file?"

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Thank you very much for your time and patient responses.

[ahanden]

For question 1, it looks like you found a bug! I'll need to release a patch to fix it. In the meantime, you can get the same effect by using --max-editing-nucleotides 1.

For question 2, the splicing file is not BED format. is space delimited and contains four columns:

1. Chromsome
2. Location (SAM coordinate)
3. Strand (+ or -)
4. Splice (D or A)

There are no filename conventions for the span file either.

[1127yangyuping]

When I run the program using the parameters given above, I still encounter errors. Is there a problem? Or is there a problem with the format of my splicing.txt file?

[ahanden]

I can't say for sure, but I think that either your splicing file is misformatted, or there is a bug on our end.

Can you post the first few lines of your splicing file, and also re-run reditools with the --debug option and post the stack trace?