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%).
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%).
Comment