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Old 05-12-2013, 10:08 PM   #1
vivienne_lovely
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Default The low mapping rate

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

Last edited by vivienne_lovely; 05-16-2013 at 01:34 AM. Reason: add some information
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Old 06-05-2013, 07:42 AM   #2
emolinari
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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
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Old 06-05-2013, 08:47 AM   #3
Wallysb01
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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?
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Old 06-05-2013, 06:05 PM   #4
vivienne_lovely
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Manu, thank you very much. I will try to BLASTed my unmapped reads.
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Old 06-05-2013, 06:06 PM   #5
vivienne_lovely
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Quote:
Originally Posted by emolinari View Post
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.
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Old 06-05-2013, 06:11 PM   #6
vivienne_lovely
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Smile

Quote:
Originally Posted by Wallysb01 View Post
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
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Old 06-05-2013, 06:19 PM   #7
snetmcom
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Have you taken the unaligned reads and try to map them?
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Old 06-05-2013, 06:45 PM   #8
dstorey
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It also matters how closely related your sample is to a reference genome and the settings you used for mapping.
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