16S Library Problems - Help!

[joze]

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

[SNPsaurus]

Do you think the first round PCR primers could still be present and shortening the amplicons made by the second round primers?

[joze]

I have considered this previously. I did initially carry out a clean-up after the first round, but it was adversely affecting the final construct yield.

Since posting my initial question, I re-ran the reactions, increasing the number of cycles in the Round 2 PCR. I have re-submitted the samples for QC and (hopefully!) sequencing, so will update with the result.

Thanks for your suggestion,

Joze

[SNPsaurus]

I think your lower yield is a sign that the first round PCRs were contributing to the yield when not cleaned up. Additional PCR cycles should help if the second round primers are in excess and have a favorable Tm. The first round products won't bind to the flowcell so having some portion of them in the mix will only make quantifying more difficult but shouldn't mess up the good fragments. Good luck!

[joze]

Thanks very much for your help! Much appreciated. Fingers crossed for the QC today...!

[sara_ahmed]

Were the primers HPLC purified? There are often truncated sequences that come along with regular desalt primers and that might not be giving you the correct adapter ends.

Sara Ahmed, PhD | Director of Sequencing | Cofactor Genomics
3141 Olive St. | St. Louis, MO 63103 | tel. (314) 531-4647

[joze]

Dear Sara,

Yes, the primers were purified by HPLC. I was warned that desalting wouldn't be up to scratch.

Thanks,

Joze