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Old 12-21-2013, 11:10 PM   #6
kcchan
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Location: USA

Join Date: Jul 2012
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Yep you got it. The adapter sequences themselves are this way by necessity rather than by design. The Nextera enzyme inserts a specific transposed sequence of DNA during the tagmentation process, which forces you to use this sequences as your primer. The way directionality is done is by having two different sets of enzymes charged with different 5' overhangs on the transposed sequence. During PCR the primers enrich only fragments with different overhangs at each end and also add the Illumina adapter primers sequences to both ends.

The story is a bit different with TruSeq adapters. Here the identical sequence is necessary to form the double stranded sequence necessary for adapter ligation. 5' and 3' overhangs are used to add the full Illumina adapter sequence at each end. This improves efficiency over having two separate adapters as all ligated DNA form sequencing compatible product on both DNA strands, so the theoretical efficiency of the TruSeq adapters is 4x that of having two separate double stranded adapters.

Last edited by kcchan; 12-21-2013 at 11:18 PM.
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