Hi all,
I have a question about using custom primers to sequence 16S rRNA using MiSeq. I have not done a run since last April and while reviewing updated manuals from Illumina today came across a troubling statement. This text was added in July 2013, and is completely new to me.
"When custom primers are used for Read 1 and Read 2, the software directs the sippers to reservoirs 18 and 20; therefore, Illumina primers are not used for the sequencing run. If Illumina primers are not used, the optional Illumina PhiX control cannot be sequenced."
The way it was previously explained to me is that with a low diversity library such as 16S, during a given cycle, all the clusters light up with the same color. This is why it is necessary to add PhiX which will provide diversity. If this is the case then PhiX needs to be sequenced simultaneously with the library. I thought that Illumina sequencing primers that are a part of the kit, get injected into the flow cell together with the custom primers that I load, which allows for PhiX sequencing.
Has anyone done 16S sequencing on MiSeq recently? Is this new to v3 kits?
Thanks in advance!
I have a question about using custom primers to sequence 16S rRNA using MiSeq. I have not done a run since last April and while reviewing updated manuals from Illumina today came across a troubling statement. This text was added in July 2013, and is completely new to me.
"When custom primers are used for Read 1 and Read 2, the software directs the sippers to reservoirs 18 and 20; therefore, Illumina primers are not used for the sequencing run. If Illumina primers are not used, the optional Illumina PhiX control cannot be sequenced."
The way it was previously explained to me is that with a low diversity library such as 16S, during a given cycle, all the clusters light up with the same color. This is why it is necessary to add PhiX which will provide diversity. If this is the case then PhiX needs to be sequenced simultaneously with the library. I thought that Illumina sequencing primers that are a part of the kit, get injected into the flow cell together with the custom primers that I load, which allows for PhiX sequencing.
Has anyone done 16S sequencing on MiSeq recently? Is this new to v3 kits?
Thanks in advance!
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