48 v 96 multiplexing

[devorevo]

I have approximately 200 samples I'm planning to sequence. I originally planed on multiplexing (Illumina Nextera XT) 96 samples to make two libraries, and then sequence a library per lane for a total of two lanes. I talked to someone in our core facility about the plan and they recommended we multiplex to a maximum of 46 samples (which would then require 4 lanes total...doubling our sequencing costs) due to the possibility of some samples becoming over/underrepresented in number of reads.

I talked with a rep from Illumina and they said multiplexing to 96 should be fine as long as the initial sample inputs amounts are the same. We quantify our samples with Qubit HS.

Any experience or advice on multiplexing 48 v 96?

[fanli]

Depends how much sequencing depth you need per sample..

[devorevo]

Depends how much sequencing depth you need per sample..

We're looking at a minimum of ~1M reads per sample and we plan on using a system that will generate ~330M reads per lane. Because of our lower minimum threshold, we're not too concerned if the average read is 3.4M v 6.8M.

[fanli]

I'd be okay with the 96-plex then. You can always repool and sequence a third run if some samples need more depth.

For reference, attached is the indexing breakdown we got recently from a pool of ~130 Nextera XT libraries.