sequencing rRNA with PGM

[vbiaudet]

Hi,

I analyze data coming from Ion PGM RNAseq and I found 20% of reads that correspond to rRNA even if the library was constructed to extract mRNA (polyA). Did somebody detect the same problem with PGM prep library?

Thank you, Véronique

[gcristof]

Is the rRNA from the same organism as your RNA-seq library? Bacterial rRNA is a common contaminant of some molecular biology reagents. Could it be the reason?

[IonTorrent]

Hi Véronique,

What method of poly(A) enrichment did you use? Is this a human RNA sample? What you describe generally has its roots in the initial RNA enrichment/depletion, as opposed to the library preparation itself. In our experience, one can achieve <5% rRNA in the final library when starting with a good quality sample with mRNA enrichment. For example, we recommend using the Dynabeads mRNA DIRECT Micro Kit or the MicroPoly(A) Purist Kit from Life Technologies, depending on the amount of starting material that you have.

Alternatively, for whole transcriptome sequencing you could consider employing ribosomal RNA depletion, for example using the Ribominus Eukaryote Kit v2 also from Life Technologies.

There's a wealth of RNA-Seq expertise here on SEQanswers so I'm sure you'll find plenty of support here. Your Life Technologies Field Applications Scientist can also be helpful with respect to the PGM RNA-Seq library preparation itself. Lastly, I'd recommend checking out the Ion Community, specifically the main PGM discussion forum or the RNA-Seq Applications space.

Originally Posted by vbiaudet

Hi,

I analyze data coming from Ion PGM RNAseq and I found 20% of reads that correspond to rRNA even if the library was constructed to extract mRNA (polyA). Did somebody detect the same problem with PGM prep library?

Thank you, Véronique

[vbiaudet]

It's the same organism....

Originally Posted by gcristof

Is the rRNA from the same organism as your RNA-seq library? Bacterial rRNA is a common contaminant of some molecular biology reagents. Could it be the reason?

[jonathanjacobs]

I doubt this would be a problem specific to the PGM.

If you are just looking at mature eukaryotic mRNAs, and have plenty of starting material, then using a two step process works very well 1) deplete rRNA first, and then 2) poly(A) pull down.

Also - keep in mind not all poly(A) kits are created equal; and not all rRNA depletion kits work well for all euks...

[vbiaudet]

Yes it's not specific of PGM but depend of the kit that is specific to the sequencer used, I have not the same problem with sequences coming from Illumina (same sample).

I have also a problem of chimeric reads, for me a chimeric read is for a read of 100 bases length, from 1 to 50 the read match perfectly gene1 and the same read from 51 to 100 match perfectly gene2, gene2 being very different from gene1, other location...
Someone know a tool to detect or count chimeric reads from a sam file?

Thank you,

Originally Posted by jonathanjacobs

I doubt this would be a problem specific to the PGM.

If you are just looking at mature eukaryotic mRNAs, and have plenty of starting material, then using a two step process works very well 1) deplete rRNA first, and then 2) poly(A) pull down.

Also - keep in mind not all poly(A) kits are created equal; and not all rRNA depletion kits work well for all euks...

[pmiguel]

Originally Posted by vbiaudet

Yes it's not specific of PGM but depend of the kit that is specific to the sequencer used, I have not the same problem with sequences coming from Illumina (same sample).

Illumina's (included) polyA reagents work better than any other kit we have ever tried. Nothing else even comes close.

I don't know if any other library construction kit includes the poly+ RNA purification step. If yours does not, then typically we would need to do 2 cycles of oligo dT binding step to effectively deplete the sample of rRNA.

To understand why this should be necessary, check out this paper. Specifically this section from its text:

Supplementary Note 4. Difference between actual and observed rRNA removal
The observed post-depletion rRNA fraction does not accurately reflect the amount of rRNA that is actually removed. To illustrate this, let us assume a hypothetical total RNA sample containing 95 rRNA and 5 mRNA molecules (i.e. 95% observed rRNA). Removal of 80% of the rRNA leaves 19 rRNA molecules and the original 5 mRNAs (i.e. 79% observed rRNA). This demonstrates that the observed rRNA depletion is only 16% despite 80% actual removal.

--
Phillip

[RonanC]

I am doing RNA-seq on bacterial RNA samples and I'm having terrible problems with rRNA contamination. Up to 90% of my reads are rRNA!

Currently I am using the Ambion MicrobExpress kit to remove rRNA (from S. aureus) but clearly it is not working. Does anyone else have any experience with bacterial RNA-seq? What rRNA removal kit are you using and what % of your reads are ribosomal?

[Gorbenzer]

anyone have experience with bacteria??

[Tucker303]

Originally Posted by RonanC

I am doing RNA-seq on bacterial RNA samples and I'm having terrible problems with rRNA contamination. Up to 90% of my reads are rRNA!

Currently I am using the Ambion MicrobExpress kit to remove rRNA (from S. aureus) but clearly it is not working. Does anyone else have any experience with bacterial RNA-seq? What rRNA removal kit are you using and what % of your reads are ribosomal?

We are about to try the Ribo-Zero kit on S. aureus. Did any of you make any more progress on bacterial systems?