Can I pool RNA-seq library with ATAC-seq library and sequence in the same S4 lane?

Question

[cwwong13]

I have one extra lane available with 4 more ATAC-seq samples to run. I wonder whether I can pool RNA-seq library with ATAC-seq library and sequence in the same S4 lane. So I do not need to waste the sequencing capacity.

Would be nice If you can suggest how to pool the two libraries.

Thanks!

Answer 1

[GenoMax]

As long as your have compatible indexes that allow pooing and the two libraries have similar insert sizes pooling them is fine for sequencing. You should pay attention to insert sizes when making the library pool (make sure you quantitate individual libraries well). Smaller inserts will preferentially cluster and may skew final distribution of data. Number of cycles should not be a huge problem as long as they are between 50-100 bp.

[cwwong13]

Thanks for the prompt reply. My ATAC-seq is following the Omni protocol, thus I am using the Nextera adapter and indices ad2.40- ad2.43. Do you think that would be compatible with the KAPA UDI Adapter Kit? The Kit is using the Dual-Indexed Adapter, May I know which column I should refer to (They have P7 index sequence and P5 index sequence)?

[GenoMax]

You should compare the indexes for the samples/kits. Depending on how other three lanes on your S4 are running you may be forced to run with 1-D or 2-D indexes. If indexes with 1-D overlap then you will have to omit at least samples that overlap. With 2-D you may have a better chance of getting all samples through. If there are any overlaps then you will simply lose the data for those samples so don't pool them in first place.