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!
Cheers,
Joze
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!
Cheers,
Joze