The low mapping rate

[vivienne_lovely]

Hi all,
I just got some RNA-seq data and mapped them to hg19 by tophat, the RNA enrichment was based on RiboMinus rRNA depletion,
but the mapping rate is just 41%. How to explain this? What should I do with those unmapped reads next? I am really new in this field, would you please help me? Any reply will be appreciate.
Vivienne

[emolinari]

Hi Vivienne,

I am experiencing the same issue. I have BLASTed my unmapped reads and I can clearly see that there is a lot of rRNA contamination.
You can try to do the same, I have realized that is quite common in ribo depleted samples!
Manu

[Wallysb01]

Ribodepleted RNAseq still have substantial rRNA, its just a lot less than without it. Anyway, that shouldn't effect the mappability for the reads.

Vivienne, have you run fastqc or fastx quality stats on your reads to try to see what might be going on with the reads themselves? They might not be mapping due to low quality, base pair biases, or adapter contamination. In some cases trimming could help the mappability.

Also, from what kind of sample did you do an RNA extraction? Is it possible there is a lot of microbial RNA being sequenced?

[vivienne_lovely]

Manu, thank you very much. I will try to BLASTed my unmapped reads.

[vivienne_lovely]

Hi Vivienne,

I am experiencing the same issue. I have BLASTed my unmapped reads and I can clearly see that there is a lot of rRNA contamination.
You can try to do the same, I have realized that is quite common in ribo depleted samples!
Manu

Thank you, Manu, I wil try to BLASTed my unmapped reads.

[vivienne_lovely]

Ribodepleted RNAseq still have substantial rRNA, its just a lot less than without it. Anyway, that shouldn't effect the mappability for the reads.

Vivienne, have you run fastqc or fastx quality stats on your reads to try to see what might be going on with the reads themselves? They might not be mapping due to low quality, base pair biases, or adapter contamination. In some cases trimming could help the mappability.

Also, from what kind of sample did you do an RNA extraction? Is it possible there is a lot of microbial RNA being sequenced?

Hi, Wallysb01
I already run the fastqc, and I also filtered the low quality reads and trimmed adapter contaminations. But I did not pay attention to the microbial RNA. Thank you for your reply, it really helps a lot.
Vivienne

[snetmcom]

Have you taken the unaligned reads and try to map them?

[dstorey]

It also matters how closely related your sample is to a reference genome and the settings you used for mapping.