2nd round miseq

[apolanow]

Hi
I am wondering if you can help me please.
My boss has recently returned from a metabarcoding conference in Europe and noted that labs are now using dual indexes in the first round of PCR and then sending their library away to have the second round ligated on for miseq.
We are going to try and do this in house. We are thinking of dual barcoding the first round of PCR with 6bp indexes. We would then like to either
a. Use a prep that ligates Illumina adapters onto the pool of amplicons
b. Do the whole reaction in one tube. Ie a 2-phase PCR addition. Some labs have got this to work, apparently. Early round PCR is at a low temp for the marker, then a second phase adds the tags and P5+P7.

We currently do a 2-step PCR addition of MIDs+P5 and P7 sequences. We use the Illumina protocol for 16S rDNA bacterial as a guide. It has problems with contamination control and 2-step PCR is very fidly, prone to pipetting errors and time consuming.

Any help with this would be appreciated.

Thanks
Andrea

[nucacidhunter]

The approach depends on your target region and the analysis type.

If the aim is to analyse and compare samples quantitatively then barcoding primers might affect the result by preferential amplification of some targets in different samples because the barcode overhang will be different in samples. One tube two step PCR would have more artefacts and possibly low yield. For this approach target size should be long enough to allow proper clean up of products from primers interactions.

Two step PCR will be labour intensive but results will be reproducible and quantitatively comparable.

If you are looking for presence-absence either of them that suits your lab set up should be fine.

[martinjf]

totally agree with the first answer.
solution a works fine but is costly and time consuming. We did it for years and moved to the 2-step pcr that is bot hvery flexible and quite cheap. The solution b works fine for particular systems (as v3-v4 16S regions for example) but needs particular fine tuning for each new marker.
I was probably at the same meeting (Upsala) and most presentations were using a 2-steps PCR approach.