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Old 08-06-2008, 08:15 PM   #1
fedora
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Default Sequencing WGA ChIP DNA

I am trying to work out good approach to sequence ChIP DNA that has been amplfied with the GenomePlex Whole Genome Amplification protocol. DNA from this seems to be mostly 400-500bp. Because of the primer sequences added to the ChIP fragments (not sure how long these are - does anyone know?) it doesnt make sense to sequence these directly with Solexa.
Has anyone tried concatenation of WGA DNA followed by shearing? Are there any other approaches that are worth trying?
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Old 08-19-2008, 06:44 AM   #2
andibody
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Primers are 58 and 34 nucleotides. Your average fragment size would be around 550. This would be OK for Solexa sequencing (some people use fragments up to 700 bp). But expect your peaks to be broader when sequencing bigger sized fragments. Nonetheless, if your antibody is highly specific it should not matter.
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Old 08-20-2008, 02:28 PM   #3
csquared
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Since your WGA material is already available, your best bet is to polish the ends using the End-It DNA Repair kit (Epicentre) and then concatenate the amplified DNA together using the Quick Ligation kit from NEB. Use 5ul ligase/ug DNA. Then re-fragment the DNA and proceed as usual with the library prep. You will need to remove the WGA primer sequences from your reads during analysis. Expect about 20% or so of the reads to be primer sequences.
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Old 08-26-2008, 06:24 AM   #4
fedora
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Thanks csquared. Have you actually tried this approach? Is there a bioinformatic problem in working out the sequence at the margins of the adapters? Eg how are reads that only have a couple of WGA adapter/primer nucleotides at the end discerned from SNPs? I'm no bioinformatician, but this seems like a potential challenge to me given the sequence coverage is theoretically random?
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Old 01-19-2011, 12:06 AM   #5
mudshark
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Did actually anyone do this already, Solexa sequencing of WGA amplified material? Would be very interested to know if it worked. How long are the Genomeplex primers? Whats the best way of avoiding sequencing of the WGA primers, ligation+fragmentation or just fragmentation after WGA?
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Old 01-20-2011, 04:17 AM   #6
OptimusBrien
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Default question for csquared...

Hey csquared,
It sounds like you have direct experience of performing Solexa sequencing of WGA material. I'm experiencing some technical issues (ChIP issues not sequencing issues) which are resulting in me having limited scope for performing sequencing on my ChIP DNA. Bottom line, it looks like performing WGA prior to sequencing is probably the only viable option I have left at this point. I'd really like to hear of your experience with this. I have several questions. Presumably you used Sigma's WGA2 kit? and the blunting/concatenation steps, are these done in line with the instructions from the 2 suppliers? Or did you have to fiddle a bit? How did you fragment the concatenated DNA? Enzyme or sonication? Thanks for any help you can provide I REALLY appreciate it!!
Optimus
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