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rRNA remove from RNAseq data

Hi everyone,

I mapped human RNAseq raw data to Refseq rRNA sequences using bwa, it turned out that 20% raw reads mapped to rRNA sequences. Is it normal? But I have used a kit to remove rRNA including cytoplasmic(5s, 5.8s, 18s, 28s) and mitochondrial(12s, 16s) ribosomal RNA. I found that the most mapped reads belong to 45S pre-ribosomal RNA sequences. I have no idea about this. Someone could help me! Any suggestion is welcome!

Thanks!

rna-seq

You could use riboPicker to identify and remove rRNA reads from the fastq files. I am not sure if SILVA databases include the pre-rRNA sequences though.

4 answers

Do you plan to assemble a transcriptome? If not, you don't have to remove them. When assigning reads to feature of a GTF, they will not be counted anyways. 20% does not sound too bad. If you did not use a kit at all, it would be like 95%, so as long as you get sufficient reads mapping on target for your analysis, I would not care about them. Still as a remark, BWA is not a splice-aware aligner. You might consider using HISAT2 or STAR for RNA-seq alignments.

This sounds like a good suggestion, it is not clear if OP is carrying out standard RNASeq analysis or something else e.g. metatranscriptomics.

thank you! I only see the rRNA rate in my fastq file using BWA for mapping to refseq rRNA sequences. I just want to know the efficiency of the kit used. I don't know why 45S pre-rRNA is not removed. 20% accounts for about 6G of data, which I think is a big waste. I want to reduce this waste. I am carrying out standard RNASeq analysis.

I have used SortMeRNA before and worked very well for me.

SortMeRNA is useful to remove rRNAs. Do you know any option to save these rRNA reads? I want to do some further analysis to these rRNAs. Thanks.

You can use bbduk.sh to filter RNA reads and save them. A Guide is here.

There is a program named BBSplit that you can use to filter and remove your undesired sequences

There are a number of tools specifically for rRNA reads removal. Recently, we also developed a rRNA reads detection method named RiboDetector (https://github.com/hzi-bifo/RiboDetector), and in the article we benchmarked different computational tools: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkac112/6533611. RiboDetector is the most computationally efficient and most accurate software for rRNA reads removal. It can be used out-of-the-box without any database:

  • GPU mode:

    ribodetector -t 20 \
    -l 100 \
    -i inputs/reads.1.fq.gz inputs/reads.2.fq.gz \
    -m 10 \
    -e rrna \
    --chunk_size 256 \
    -o outputs/reads.nonrrna.1.fq outputs/reads.nonrrna.2.fq
    
  • CPU mode

    ribodetector_cpu -t 20 \
    -l 100 \
    -i inputs/reads.1.fq.gz inputs/reads.2.fq.gz \
    -e rrna \
    --chunk_size 256 \
    -o outputs/reads.nonrrna.1.fq outputs/reads.nonrrna.2.fq
    

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