Hi everyone,

First of all, I am aware that this topic has been discussed before, however, I could not find a suitable answer for my needs.

We used NovaSeq 6000 to sequence multiple samples with different library design:

1. The first has dual indices that are 8bps long.
2. The second has one index that is also 8bps long (index 2 - i5)
3. The third has no indices, only a barcode sequence in the read itself

The reads structure in the RunInfo.xml file looks like this:

<Reads>
<Read Number="1" NumCycles="55" IsIndexedRead="N"/>
<Read Number="2" NumCycles="8" IsIndexedRead="Y"/>
<Read Number="3" NumCycles="8" IsIndexedRead="Y"/>
<Read Number="4" NumCycles="35" IsIndexedRead="N"/>
</Reads>

I think that the only possible way is to create two sample sheets, the first for the dual indices:

[Header],,,,,,,,
<some header stuff>
,,,,,,,,
[Reads],,,,,,,,
Read1Cycles,55,,,,,,,
Read2Cycles,35,,,,,,,
Index1Cycles,8,,,,,,,
Index2Cycles,8,,,,,,,
,,,,,,,,
[Settings],,,,,,,,
,,,,,,,,
[Data],,,,,,,,
Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,index2,Sample_Project,Description
sample1_with_dual_indices,,,,,CTCTCTAC,ATAGAGAG,blabla,blabla
sample2_with_dual_indices,,,,,CGAGGCTG,AGAGGATA,blabla,blabla
sample3_with_dual_indices,,,,,CGAGGCTG,CTCCTTAC,blabla,blabla
sample_n_with_dual_indices,,,,,CGAGGCTG,TATGCAGT,blabla,blabla

and using the "Undetermined" files here for the samples with no indices.

Finally, making another SampleSheet for the single index sample, same as above but using only the index2 column.

Regarding bcl2fastq arguments, should it be:

--use-bases-mask Y*,I8n*,I8n,Y*

for the dual indices SampleSheet run, and

--use-bases-mask Y*,I8n*,Y*

for the single index run.

I don't have much confidence in this strategy, but this is the best I came up with. Please let me know what do you think.

Thanks!