Illumina Genomic DNA Sample Prep

kwebb · 02-10-2009, 12:07 PM

I have run 2 GA II flowcells and on each cell only about 50% of my reads align to the reference genome. The remaining 50% of the sequences are made up of high amounts of primer sequence and trash reads. As such, we going to redo our library prep and I would like to get some information from others doing the same.

I am using the Illumina protocol "Preparing Samples for Sequencing Genomic DNA". Briefly, our protocol involves sonicating 5000ng of DNA for 30 seconds in 50ul of TE. After the Qiagen PCR purification kit clean-up we are left with only about 20% of our sample as measured by Nanodrop spec.

My questions are:
How do you fragment your DNA?
If you sonicate, how much DNA do you start with and in how much and in what buffer to you sonicate?
How much of your starting material do you recover after the Qiagen clean-up?
Per sample, how much primer/trash sequence do you get back?

Thanks in advance!

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02-10-2009, 12:07 PM	#1
kwebb Member Location: Wahington, DC Join Date: Jul 2008 Posts: 21	Illumina Genomic DNA Sample Prep I have run 2 GA II flowcells and on each cell only about 50% of my reads align to the reference genome. The remaining 50% of the sequences are made up of high amounts of primer sequence and trash reads. As such, we going to redo our library prep and I would like to get some information from others doing the same. I am using the Illumina protocol "Preparing Samples for Sequencing Genomic DNA". Briefly, our protocol involves sonicating 5000ng of DNA for 30 seconds in 50ul of TE. After the Qiagen PCR purification kit clean-up we are left with only about 20% of our sample as measured by Nanodrop spec. My questions are: How do you fragment your DNA? If you sonicate, how much DNA do you start with and in how much and in what buffer to you sonicate? How much of your starting material do you recover after the Qiagen clean-up? Per sample, how much primer/trash sequence do you get back? Thanks in advance!