Dear SEQanswers
I have a very general question and would love to have input from at least a handful of different people with experience in this field.
We are doing 16S profiling of bacteria from human/mouse host tissue samples and I find this pdf on the net that seems to be a decent starting protocol that is in use worldwide for exactly this type of study:
In the protocol, the barcode is added to the R (reverse) primer only and sequencing is done in a single direction using that R primer.
Strangely, the local sequencing center claims that they had problems with single direction sequencing and they only sequence in both directions, but not paired. This results in barcodes on both primers and compatibility issues with mothur/qiime pipelines for processing flowgrams. They even went as far to say that the new Roche kits do not do single direction sequencing.
Sadly, I do not have the experience nor the knowledge to support or refute such claims. But I am hoping that people here do and can help me arm myself for my next meeting so that I can do 454 16S sequencing in a standardize manner that is most compatible with the software that I want to use to process the output.
I have a very general question and would love to have input from at least a handful of different people with experience in this field.
We are doing 16S profiling of bacteria from human/mouse host tissue samples and I find this pdf on the net that seems to be a decent starting protocol that is in use worldwide for exactly this type of study:
In the protocol, the barcode is added to the R (reverse) primer only and sequencing is done in a single direction using that R primer.
Strangely, the local sequencing center claims that they had problems with single direction sequencing and they only sequence in both directions, but not paired. This results in barcodes on both primers and compatibility issues with mothur/qiime pipelines for processing flowgrams. They even went as far to say that the new Roche kits do not do single direction sequencing.
Sadly, I do not have the experience nor the knowledge to support or refute such claims. But I am hoping that people here do and can help me arm myself for my next meeting so that I can do 454 16S sequencing in a standardize manner that is most compatible with the software that I want to use to process the output.
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