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
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
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