Illumina stranded RNA-Seq background problem

NicoBxl · 07-01-2013, 05:38 AM

Hi everybody,

We perform RNA-Seq experiments using illumina TruSeq total RNA stranded (with RiboZero) and we have strange results compared to previous samples. We sequenced on a HiSeq2000 2x100bp.

Here a picture of the alignment results :

The two first lane are a previously sequenced sample (same kit) and the two below are a new one. You can see that on the new ones, there is a background. And it is almost on the whole genome like that...

Someone has an idea of where the problem comes from?

Thanks a lot,

N.

Bukowski · 07-01-2013, 11:31 AM

Well if it's all over the genome, I'd suggest it was genomic DNA. Contamination or sample mix-up.

Genohub · 07-06-2013, 08:37 PM

Looks like genomic DNA contamination. Did you treat your RNA with DNAse?

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07-01-2013, 05:38 AM	#1
NicoBxl not just another member Location: Belgium Join Date: Aug 2010 Posts: 264	Illumina stranded RNA-Seq background problem Hi everybody, We perform RNA-Seq experiments using illumina TruSeq total RNA stranded (with RiboZero) and we have strange results compared to previous samples. We sequenced on a HiSeq2000 2x100bp. Here a picture of the alignment results : The two first lane are a previously sequenced sample (same kit) and the two below are a new one. You can see that on the new ones, there is a background. And it is almost on the whole genome like that... Someone has an idea of where the problem comes from? Thanks a lot, N.

07-01-2013, 11:31 AM	#2
Bukowski Senior Member Location: UK Join Date: Jan 2010 Posts: 390	Well if it's all over the genome, I'd suggest it was genomic DNA. Contamination or sample mix-up.

07-06-2013, 08:37 PM	#3
Genohub Registered Vendor Location: genohub.com Join Date: Mar 2013 Posts: 210	Looks like genomic DNA contamination. Did you treat your RNA with DNAse?