Hi Everyone,
I am new to the forum and am hoping to find some help regarding primer design and sequencing for conducting 16S metagenomics studies using the Illumina MiSeq.
I have previously worked with preparing samples for and analyzing 454 data and am switching to using an Illumina MiSeq. Currently, I am interested in amplifying the V4 region of 16S and have the primer sequences targeting that region. However, the custom amplicons from Illumina generated using DesignStudio (which include their adapters) from my understanding are only able to be based on the human genome rather than bacterial. So, I am trying to understand how to go about designing the primers and linkers needed for amplifying my region of interest that will include the adapter regions needed to match those on the Illumina flow cell.
In a recent paper by Caporaso et al. (2011), there is a figure depicting the initial and sequencing primers with the forward primer having an Illumina adapter, linker, and primer sequence and the reverse having all of these along with the barcode sequence. This is what I would like to do, but I do not understand how to get the adapter sequence to match the Illumina-specific adapters located on their flow cell. I have a feeling it is just that I am new to this entire process and am missing something. So, I thought I would check with the forum to see if anyone had any suggestions.
Thank you all in advance!
I am new to the forum and am hoping to find some help regarding primer design and sequencing for conducting 16S metagenomics studies using the Illumina MiSeq.
I have previously worked with preparing samples for and analyzing 454 data and am switching to using an Illumina MiSeq. Currently, I am interested in amplifying the V4 region of 16S and have the primer sequences targeting that region. However, the custom amplicons from Illumina generated using DesignStudio (which include their adapters) from my understanding are only able to be based on the human genome rather than bacterial. So, I am trying to understand how to go about designing the primers and linkers needed for amplifying my region of interest that will include the adapter regions needed to match those on the Illumina flow cell.
In a recent paper by Caporaso et al. (2011), there is a figure depicting the initial and sequencing primers with the forward primer having an Illumina adapter, linker, and primer sequence and the reverse having all of these along with the barcode sequence. This is what I would like to do, but I do not understand how to get the adapter sequence to match the Illumina-specific adapters located on their flow cell. I have a feeling it is just that I am new to this entire process and am missing something. So, I thought I would check with the forum to see if anyone had any suggestions.
Thank you all in advance!
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