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Old 08-06-2013, 03:13 AM   #1
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Location: Edinburgh

Join Date: Oct 2012
Posts: 7
Default 16S Library Problems - Help!

Hello everyone,

I am trying to study the pig gut microbiome, utilising 16S amplicon sequencing on the MiSeq. After optimising barcoded primers for this and creating a construct at correct size (~300bp), I submitted this for QC. It has become evident that the adapter sequence is present in very low quantities (if at all) after carrying out qPCR.

In more detail, I create the construct using 2 PCR rounds. The first round are common read 1 and read 2 primers which add a section of the adapter sequences. The second PCR round extends out from this, utilising a common read 1 primer extension for all samples and a variable (barcoded) read 2 primer specific to each sample (4 cycles). I add these primers directly to the product from the round 1 PCR and carry out a further 4 cycles of PCR before cleaning up with AMpure beads.

I am confused, since a product is clearly evident on an agarose gel and also gave decent tapestation results. However, I have been told that sequencing these products will be useless, due to the very low qPCR reads. If anyone has any advice on this matter, I would be very grateful!


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