Use of two Read 1 primers on MiSeq

[FNB]

I'm planning on sending out for sequencing soon using a MiSeq PE 250. I was wondering if its possible to use two Read 1 primers at once? I have a 16s sample that contains the PCR from two different primers (a general Bac and specific). Will running the sample with a Gen Bac Read 1, Specific Read 1, and their reverse Read 2 work? Or do I need to split them up into two seperate runs? I'm afraid it may cause interference.

[kmcarr]

Originally Posted by FNB

I'm planning on sending out for sequencing soon using a MiSeq PE 250. I was wondering if its possible to use two Read 1 primers at once? I have a 16s sample that contains the PCR from two different primers (a general Bac and specific). Will running the sample with a Gen Bac Read 1, Specific Read 1, and their reverse Read 2 work? Or do I need to split them up into two seperate runs? I'm afraid it may cause interference.

As a general principle it will work. The stansard practice for 16S amplicon sequencing was to spike in a large amount of PhiX to provide base balance. Custom primers are used for the 16S while standard primers for the PhiX, demonstrating that libraries using different sequencing primers can be mixed in one MiSeq run.