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bioinfo-biols / CIRI-cookbook

Document for CIRI-series software
https://ciri-cookbook.readthedocs.io/en/latest/index.html
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CIRI2 : CIRI_AS_v1.2.pl : Please make sure the SAM is the same one used for circRNA detection by CIRI without modification! #35

Open Thananonsr opened 2 months ago

Thananonsr commented 2 months ago

Hi everyone Currently, I am analyzing circRNAs from an RNA-Seq dataset using CIRI2 tools, following the steps outlined in the CIRI-cookbook. Here's how I have proceeded:

  1. Checked the FastQC of the raw data files.
  2. Trimmed adapters using Trimmomatic and then checked the FastQC.
  3. Aligned reads using BWA-MEM to obtain a .sam file.
  4. After obtaining the trimmed_sample.sam file, I ran CIRI using the following command: perl CIRI2.pl -I trimmed_sample.sam -O sample/trimmed_sample.ciri -F bwa.index_hg38.fa/hg38.fa -A knownGene.gtf
  5. After getting output.ciri, I ran the following command: perl CIRI_AS_v1.2.pl -S trimmed_sample.sam -C sample/trimmed_sample.ciri -F bwa.index_hg38.fa/hg38.fa -A knownGene.gtf -O sample/output -D yes

However, I encountered an issue when I ran the last command. It failed with the error message: "Please make sure the SAM is the same one used for circRNA detection by CIRI without modification!"

My question is:

  1. I want to understand what the error SAM is the same one used for circRNA detection by CIRI without modification! means and how it occurred, as I haven't made any changes to the .sam file? I've tried to find a solution but haven't been able to fix it, so I would like to ask for your help.

Thank you

Kevinzjy commented 2 months ago

Hi @Thananonsr , please make sure you are running CIRI2 & CIRI-AS with paired, untrimmed reads.

Thananonsr commented 2 months ago

Hi @Thananonsr , please make sure you are running CIRI2 & CIRI-AS with paired, untrimmed reads.

Of course, I have been running CIRI2 & CIRI-AS with paired-end trimmed reads.

Kevinzjy commented 2 months ago

Yes, that's the problem. CIRI-AS cannot handle reads with different lengths after trimming, so could you try to use untrimmed reads to instread?

Thananonsr commented 2 months ago

Yes, that's the problem. CIRI-AS cannot handle reads with different lengths after trimming, so could you try to use untrimmed reads to instread?

You means use the raw data as a input?

Kevinzjy commented 2 months ago

You means use the raw data as a input?

Yes, only raw data is supported.

Thananonsr commented 1 month ago

Thank you, sir. But now I have been running the same step in another dataset (e.g. GSE147009 *RNA-seq data) with raw data. However, the raw data from this database has already had the adapters removed from all samples (24 samples). When I ran the following command: perl CIRI_AS_v1.2.pl \ -S /home/storeone/sirinar/thananon/SRR11310298/SRR11310298.sam \ -C /home/storeone/sirinar/thananon/SRR11310298/SRR11310298.ciri \ -F bwa.index_hg38.fa/hg38.fa \ -A gencode.v46.annotation.gtf \ -O /home/storeone/sirinar/thananon/SRR11310298 \ -D yes I reencountered the same issue: 'Please make sure the SAM is the same one used for circRNA detection by CIRI without modification!'

Q: Do you have any suggestions for this issue? I need help since I can't find this database with untrimmed adapters.

Thananonsr commented 1 month ago

And here are all the steps in my analysis:

  1. Raw data (RNA-seq)
  2. FastQC
  3. Index human reference genome
  4. Align reads of the sample with BWA MEM -T 19
  5. Run perl CIRI2.pl to detect circRNAs
  6. Run perl CIRI_AS
  7. Running the CIRI-full pipeline
  8. Running CIRI-vis
  9. circRNA quantification (CIRIquant)

I followed the above steps for circRNA analysis per sample, and the resulting data is in GTF format.

--> I’m wondering if it's necessary to run all the steps from 5-9. I’ve already obtained circRNA data from step 5, but I’m unable to run step 6. Q: 1. Can I take the circRNA data from step 5 and skip directly to step 9? Or would this negatively affect the circRNA data output?****

Kevinzjy commented 1 month ago

Thank you, sir. But now I have been running the same step in another dataset (e.g. GSE147009 *RNA-seq data) with raw data. However, the raw data from this database has already had the adapters removed from all samples (24 samples). When I ran the following command: perl CIRI_AS_v1.2.pl -S /home/storeone/sirinar/thananon/SRR11310298/SRR11310298.sam -C /home/storeone/sirinar/thananon/SRR11310298/SRR11310298.ciri -F bwa.index_hg38.fa/hg38.fa -A gencode.v46.annotation.gtf -O /home/storeone/sirinar/thananon/SRR11310298 -D yes I reencountered the same issue: 'Please make sure the SAM is the same one used for circRNA detection by CIRI without modification!'

Q: Do you have any suggestions for this issue? I need help since I can't find this database with untrimmed adapters.

Hi @Thananonsr, you can trim the reads to a uniform length (e.g. 75/100bp) to run CIRI-full.