low efficiency of DSN normalization

[nasobema]

We have set up a protocol for RNA-seq based on adapter-ligation to RNA-fragments. For ribosome removal, I would like to use the DSN normalization method, which is said to work better than bead-based methods (according to Illumina).

The problem is that the efficiency seems to be very low. Our genomics department is performing the normalization for us prior to sequencing the libraries on an Illumina GA-II. I mapped the results to the reference genome and found that still on average 90 - 95 % of the reads map to ribosomal loci. Untreated samples typically have 98-99%. Thus, it's working but not satisfyingly.

The sequence is a large bacterial genome (6.2 Mb).

Does anyone have experience with DSN and could give us a hint on how to improve the efficiency?
What are the ribosome ratios that you can typically achieve with the protocol (Illumina states ~15%).

[mnkyboy]

Do you have an accurate measurement of the ng mass of the library you are DSN treating? I have been doing a lot of DSN (granted with mammalian) and I find that you really need to be in that 80-100 ng range measured by picogreen.

I still see probably 1/10 that fail but that is most likely due to me losing the CoT during treatment.

[nasobema]

Do you have an accurate measurement of the ng mass of the library you are DSN treating? I have been doing a lot of DSN (granted with mammalian) and I find that you really need to be in that 80-100 ng range measured by picogreen.

I still see probably 1/10 that fail but that is most likely due to me losing the CoT during treatment.

Yes, DNA concentration was measured with Qubit, which should be reliable.

Any more ideas?

We have been tightly following the Illumina protocol for DSN. Losing a constant temperate may always be an explanation but the technicians performing the protocol are well aware of this issue.

[irit]

A temperature drop should have the reverse effect - digestion of secondary structures.

Have you tested the enzyme to see if it is still active? Is it possible your incubation temperature is too high?

[mnkyboy]

Run the provided control with the protocol they have and that will tell you if your enzyme is still working. It is a short protocol and you can check results on gel or BioAnalyzer.

[nasobema]

A temperature drop should have the reverse effect - digestion of secondary structures.

Have you tested the enzyme to see if it is still active? Is it possible your incubation temperature is too high?

Yes, the enzyme works fine on control dsDNA.

Incubation temperature was 68° according to the Illumina protocol.
So, could it help to reduce Temperature by a few degrees?
We have currently no idea on the effect of altering the parameters like incubation Temp./Time and Hybridization Temp./Time.
We are doing some experiments on that but I would still highly appreciate input from experienced DSN users.
Thank you all for the previous replies.

[QTLdum]

We encountered very similar problems (low efficiency of normalization) when using bovine samples. Thus, we decreased the hybridization and incubation temperature from 68° to 66° and 64° C, but this did not improve normalization. We had also confirmed that the DSN worked by performing the test protocol

[monad]

We worked with more than 8,000 library prep until today, so we are not inexperienced users here for Illumina library prep.

However, DSN is not the way to go if you want to remove ribosomal RNA!

DSN works beautifully for small amount of input total RNA, even below picogreen detection level, but not an effective method to remove ribosomal RNA. Typically you will expect to have >60% of your reads are aligned to rRNA sequences.

Go with ribo minus or ribo zero type of removal methods.