Can I mix different index sizes in the same run in NovaSeq?

[Mary_R]

Hi all,

I want to mix different index sizes in the same lane in NovaSeq 6000. Is this possible?
Basically I have libraries where I used the Kapa Hyper prep protocol with Truseq dual index adapters and other older libraries where I used Meyer and Kircher protocol also with dual index. The Truseq indexes are 8bp long and the Meyer indexes are 7bp long. I want to pool these libraries made with different protocols and run them together. Is this a problem during sequencing? Can I deal with demultiplexing different index sizes after the run?
Thank you so much for your time

[luc]

Demultiplexing always happens after the run. Thus you can have several attempts if you have the software installed locally. Otherwise, you are relying on the aid of your core lab.

There are two ways to do this. The easiest way would be to demultiplex only using the first seven bases of all barcodes; you would have to check if these can discriminate all samples.

Alternatively, you could extend the Meyer and Kircher barcodes in the sample sheet by adding the subsequent base in the adapter sequence. This will likely be always the same base for the M&K barcodes.