Question: rRNA detection (for contamination) in RNA-seq
0
gravatar for Ron
3.0 years ago by
Ron970
United States
Ron970 wrote:

Hi all,

I am working with human RNA-seq data.I want to know whats the best way to get information on rRNA reads in RNA-seq? Does rRNA gets passed on in the bam files after alignment(using human genome) or we have to check the raw fastq reads only for this?

Also I want to look for this irrespective of the library they came from (polyA or rRNA removal kits),as I just want to have an estimate of this rRNA reads as a QC measure.

let me know your thoughts.

Thanks,

Ron

rna-seq star alignment next-gen • 3.7k views
ADD COMMENTlink modified 23 months ago by Charles Plessy2.7k • written 3.0 years ago by Ron970
2
gravatar for michael.ante
23 months ago by
michael.ante3.4k
Austria/Vienna
michael.ante3.4k wrote:

The rRNA-cluster are distributed in repeats over the genome. Therefore, reads origin from rRNA-molecules map too many times and get discarded by most RNA-SEQ aligners.

I'd download the rRNA fasta files from NCBI and map all reads against them (e.g. BBmap, bowtie2); the mapping rate is equal to your rRNA contamination. Additionally, you can use this as a first step to sort these reads before continuing your pipeline.

ADD COMMENTlink written 23 months ago by michael.ante3.4k
1
gravatar for igor
3.0 years ago by
igor8.3k
United States
igor8.3k wrote:

I asked a related question recently that might be useful: RNA-seq rRNA contamination

ADD COMMENTlink modified 3.0 years ago • written 3.0 years ago by igor8.3k
1
gravatar for nick.a.rouse
23 months ago by
nick.a.rouse30
nick.a.rouse30 wrote:

A very simple approach would be to download rRNA bed coordinates (from Ensembl) and then count the total number of reads that fall into this ROI (using bedtools suite) and divide this by total number of reads in the bam (samtools idxstats). This would give you a rough estimate of your background rRNA levels.

ADD COMMENTlink written 23 months ago by nick.a.rouse30
1
gravatar for Ron
23 months ago by
Ron970
United States
Ron970 wrote:

I used RseQC for rRNA detection. They have a bed file too.

http://rseqc.sourceforge.net/

ADD COMMENTlink written 23 months ago by Ron970
0
gravatar for Charles Plessy
23 months ago by
Charles Plessy2.7k
Japan
Charles Plessy2.7k wrote:

You can count or filter out rRNA reads from FASTQ files (single- or paired-end) using TagDust (and do many other things).

ADD COMMENTlink modified 23 months ago • written 23 months ago by Charles Plessy2.7k
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