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Old 08-18-2012, 04:09 PM   #32
carmeyeii
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Location: Mexico

Join Date: Mar 2011
Posts: 137
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Hello everybody,

I have a VERY basic question about the PCR step for the library preparation but I just can't seem to find the answer in my head nor in the literature. Any help is greatly appreciated.

I understand that the reason for doing an amplification step after the adapter ligation is to enrich your sample for adapter-ligated fragments which is what will actually bind to the flowcell oligos - however, doesn't this produce multiple exact copies of fragments, causing a significant amount of duplicate reads, i.e., the exact same fragment in multiple clusters on the flowcell? I suspect I may be missing out on something very basic, pergaps it is very unlikely that two or more copies of fragment x will actually make it to the hybridization step with the oligos on the flow-cell? I just don't see how having multiple clusters of a given fragment would not be a complete waste of computing power and money...

Thanks a lot!

Carmen
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