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Old 09-30-2019, 06:34 AM   #3
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Location: Purdue University, West Lafayette, Indiana

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[Note added after writing post: this exact same question, using the same wording was posted previously:


Right, you just put the index sequences in the sample sheet and the index read(s) will be that long.

Keep in mind that MiSeq cassettes are only provisioned for 2 eight base indexes + the insert reads. So if you want 25 base index reads you will be 50-16=34 cycles over the total number of cycles in the cassette if you try to run the full number of cycles.

That is, if you have a 300 cycle cassette, enough reagents are provided to do 302 + 16 cycles = 318. (Ignoring the dark cycles needed for i5 indexing...) So if you want 50 cycles of indexes, the extra 34 cycles has to come out of your insert reads. So instead of 302 cycles, you would have 276 cycles to split between your two insert reads.

I should note that I haven't ever tried to do this, I'm just going from first principles. So there might be issues. For example the MiSeq might have a maximum index length that I'm unaware of.

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