demultiplexing with same indices across different lanes

Question

[lait]

When demultiplexing, is it a problem if same indices are used across different lanes?

For example:

• lane 1 has 4 samples with indices A1, A2, A3 and A4

• lane 2 has 4 samples with indices A1, A2, A3 and A4

As far as I know, demultiplexing is done per lane? so there should be no problem in this?

Answer 1

[GenoMax]

That is correct. There should be no problem. I assume you are referring to the same indexes being used for different samples.

Note: This will not work with NovaSeq (without XP add-on) or NextSeq series of sequencers. Even though there are optically separate lanes on FC for these sequencers they are fluidically fed by the same sample pool across the entire FC.

[lait]

yes correct, different samples across different lanes having same index. thanks for your answer!

[GenoMax]

Please see the Note I added.

[lait]

we are using hiseq, I assume there should be no problem then

[GenoMax]

Correct.

[lait]

Just one follow up question, should I do a separate demultiplexing sheet for each lane in this case? or is it just one demultiplexing sheet (containing all lanes info, sample names, and indices used) fed to bcl2fastqc, and bcl2fastqc should handle the rest?

[GenoMax]

One SampleSheet.csv will be fine. Just make sure you list the samples correctly along with the lane numbers.