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Old 01-24-2014, 05:33 AM   #1
Location: Italy

Join Date: Oct 2012
Posts: 10
Unhappy Ovation Ultralow Methyl-Seq Library Systems and Miseq sequencing

Hi everyone!!
I hope that someone can help me to solve my sequencing problem.
We prepared 2 libreries with the Ovation Ultralow Methyl-Seq Library Systems and we tried to sequence them on the Miseq (100 PE).
For the read1 we used the Nugen custom primer mixed 50:50 with Illumina primer read1 mix as suggested in the library protocol while for the index read and for the read2 we used the standard Illumina primer.
Unfortunately the phix that I spiked in was only at 1% instead of 20% but I don't understand why the read1 has a high quality Q30=93% while the index read and the read 2 have bad quality (Q30=56% and 60%respectively).
The read 1 has few C while the read2 has few G suggesting that the bisulfite conversion is ok.
Nucleotides are not balanced because of the lack of C and G, and I know I need to add more phix but why that thing give me problems only in the index read and in the read2?
Many thanks for your help,
chiaraf is offline   Reply With Quote