Hello,
Our lab has just recently sequenced 16S DNA using the MiSeq platform. We had previously sequenced using the GAIIx platform and were hoping to save money by using the same primers and primer set up for MiSeq. We asked Illumina if these primers would be OK to use and they said yes, so we went forward. Now as I am attempting to process the MiSeq data I find that there are problems within the sequence that lead to me losing a lot of sequences before I am able to align them. I am currently using the mothur pipeline. I would like to also note that we used single barcodes only on the forward primer and NOT on the reverse primer (I have seen most MiSeq users using dual barcodes, could our use of only one barcode on the forward read cause any potential issues?). We are also sequencing the V1-V2 region, which I have read is not the best region to sequence with MiSeq but I still do not believe that we should be seeing as many issues with our data just because of the region.
Has anyone tried to sequence on MiSeq using a GAIIx primer set up and have you ever have you seen any problems related to your data?
Any suggestions on potential issues and solutions usign this particular method would be greatly appreciated.
here is the GAIIx forward primer sequence order:
Illumina adapter - linker - barcode - Illumina Sequencing - 16S primer
(reverse primer is the same order but lacking the barcode)
Thank you very much for your input, it is greatly appreciated!
Our lab has just recently sequenced 16S DNA using the MiSeq platform. We had previously sequenced using the GAIIx platform and were hoping to save money by using the same primers and primer set up for MiSeq. We asked Illumina if these primers would be OK to use and they said yes, so we went forward. Now as I am attempting to process the MiSeq data I find that there are problems within the sequence that lead to me losing a lot of sequences before I am able to align them. I am currently using the mothur pipeline. I would like to also note that we used single barcodes only on the forward primer and NOT on the reverse primer (I have seen most MiSeq users using dual barcodes, could our use of only one barcode on the forward read cause any potential issues?). We are also sequencing the V1-V2 region, which I have read is not the best region to sequence with MiSeq but I still do not believe that we should be seeing as many issues with our data just because of the region.
Has anyone tried to sequence on MiSeq using a GAIIx primer set up and have you ever have you seen any problems related to your data?
Any suggestions on potential issues and solutions usign this particular method would be greatly appreciated.
here is the GAIIx forward primer sequence order:
Illumina adapter - linker - barcode - Illumina Sequencing - 16S primer
(reverse primer is the same order but lacking the barcode)
Thank you very much for your input, it is greatly appreciated!
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