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Old 05-24-2011, 06:02 AM   #1
Nak
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Location: southeast europe

Join Date: May 2011
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Default DNA isolation & library quality control

Hi,
I am a beginner in high throughput sequencing so I need help with some basic questions about sample preparation (for a bacterial resequencing project on Illumina HiSeq platform, 30x coverage)

1. I have seen somewhere that the usual kits for isolation of genomic DNA are not recommended. Can you tell me why this is the case? If it is about the DNA size, why does this matter if DNA is anyway to be sheared into small pieces for sequencing?

2. What is a library quality control and is it usual to have one?

Thanks!
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Old 05-25-2011, 07:21 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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I can't think of any reason not to use "the usual kits for isolation of genomic DNA". We have not made many Illumina libraries yet, but the 4 bacterial ones we made with the TruSeq DNA library kit worked fine. We did not ask that the people submitting the DNA take any special precautions.

I would recommend actually removing the RNA from the DNA, but I'm not sure even that is important.

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Old 05-25-2011, 12:39 PM   #3
NextGenSeq
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Always do library quality control. We run an Agilent high sensitivity DNA chip and QPCR on every library. The Bioanalyzer is used to check for primer artifacts and the QPCR to quantify and check amplification efficiency.
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