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Old 03-28-2013, 01:16 PM   #1
dummyseq
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Default Nextera Exome Enrichment kit

Hi,Hello,

I am using 150ng FFPE DNA to prepare a library for HiSeq. After 1st amplification, DNA concentrations are too low, not enough for 500ng suggested for hybridisation. Followed the protocol excatly.

Any advise appreciated. Should I increase PCr cycles from 10 to 12 ?? But with Sure Select Agilent kits we haev seen that increasing PCr cycles decreases conc of DNA.

Thanks
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Old 03-28-2013, 08:38 PM   #2
epistatic
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How well did the tagmentation work with FFPE samples? You may have many fragments with one integration site, not allowing for the majority of your sample to have both adapters for the PCR step.

When I have used nextera in the past with amplicons I had to pad the ends to allow for integration space and still sequence the region of interest. I have processed 48 samples with cell line DNA with nextera exome and a bit skeptical it would work well with FFPE.

For FFPE exome I prefer a standard library followed by capture. I usually don't have to shear the DNA very much if at all before making a library.
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Old 03-29-2013, 10:06 AM   #3
dummyseq
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How exactly did you pad the ends??

Thanks
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Old 03-29-2013, 02:28 PM   #4
epistatic
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Apologies for the unclear terminology. I should have said I made the amplicon larger. If I just used the standard amplicon nextera would integrate often only once or if twice it would cut off parts that I wanted to sequence. Too small of amplicons didn't work at all with nextera.
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Old 03-30-2013, 06:28 PM   #5
snetmcom
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I would agree that Nextera with FFPE is not a good fit. You may see a reduced on target as well. Traditional library methods will likely yield better results as epistatic stated.
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Old 04-01-2013, 02:57 PM   #6
dummyseq
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Has anyone tried increasing TDE for more than 150ng DNA input? We are getting low values even after increasing input DNA and PCr cycles to 12

Any input with FFPE DNA Nextera protocol will be helpful

THanks
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Old 04-02-2013, 07:04 PM   #7
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Has this kit been renamed to "Nextera Rapid Capture Exome and Expanded Exome Kits"?
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Old 04-03-2013, 08:12 AM   #8
dummyseq
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None of the above. It just says "Nextera Exome Enrichment " Kit divided into 3 boxes,all labelled the same
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Old 10-03-2013, 06:46 AM   #9
dgasx8
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Quote:
Originally Posted by dummyseq View Post
Has anyone tried increasing TDE for more than 150ng DNA input? We are getting low values even after increasing input DNA and PCr cycles to 12

Any input with FFPE DNA Nextera protocol will be helpful

THanks
Hi dummyseq,
what about your libraries from FFPE samples? Did you obtain and captured whole exome libraries good for Hiseq?
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