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-   -   Ovation Ultralow Methyl-Seq Library Systems and Miseq sequencing (http://seqanswers.com/forums/showthread.php?t=40241)

chiaraf 01-24-2014 04:33 AM

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,
Chiara


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