rRNA removal using SortMeRNA

Question

[KMS]

Dear All,

I am analyzing microbial RNASeq data, and after aligning through bowtie2, feature counts results only RNA reads, I am unable to run SortMeRNA . Please make me understand what does it mean how to run this command I found in manual.

./indexdb_rna--ref db.fasta,db.idx [OPTIONS]

My bacterium data is not present n SIVA database but I have rRNA file of organisms from NCBI.

I also tried web based user friendly tools like rRNAfilter and RNA detector but for a beginner like me it is not "user friendly" at all. They produced only frustration but not results. (for rRNAfilter kanalyse JAR file not running and RNAdetector need a docker of which I have no idea)

I request you if any can help in any of this either SortMeRNA or any web source please comment. Your suggestions are appreciated

Thanks in advance.

[GenoMax]

Are you simply looking to see if your data has rRNA reads or do you want to clean your data removing those reads?

If that is the case you could use bbduk.sh from BBTools (are you able to work on the command line?) in filter mode like follows.

bbduk.sh -Xmx2g in=your_fastq.gz ref=your_RNA.fasta outm=reads_that_match_rRNA.fastq.gz k=25

Any reads that match rRNA sequence will be removed into reads_that_match_rRNA.fastq.gz file.

Following variation will give you cleaned data in clean.fastq.gz file.

bbduk.sh -Xmx2g in=your_fastq.gz ref=your_RNA.fasta out=clean.fastq.gz outm=reads_that_match_rRNA.fastq.gz k=25

A guide for bbduk.sh is available: https://jgi.doe.gov/data-and-tools/software-tools/bbtools/bb-tools-user-guide/bbduk-guide/

[紫枫]

Thank for your advice. But the results I tried this method were problematic.

I handle my fastq file to run this program, I use the https://www.ncbi.nlm.nih.gov/nuccore/BK000964 give rRNA fasta files as a reference. As a result, my reads_that_match_rRNA.fastq.gz reached 90% of the total reads.

It doesn't make sense. So I use blastn to process these sequences. It turns out that only 10% of these so-called "reads_that_match_rRNAs" are ribosomes

can you tell me why? Thanks a lot. my email is: [e-mail]

[KMS]

Dear GenoMax Thanks for kind and quick reply

Yes, I want to clean rRNA reads, because I used FASTQC, Trimmomatic, Bowtie2, and featurecount, but after feature count I am getting only rRNA read, only rRNA reads, don't know why. I tried everything but tired of. So I want to remove rRNA reads initially. I also tried SortMeRNA in galaxy but still getting only RNA reads.

I can work in command line in ubuntu, but don't have experience in shell .sh, but I will try to follow your suggestion. Thanks again for your response and given link.