Confusion setting up MiSeq libraries/runs

[Vanschkn]

I have previous experience setting up sequencing on a 3730 so a little confused about setting up libraries and runs on a MiSeq. So I see that you can prep libraries for 96 samples using the Nextera kits and you can even include all 96 samples in a single run. If I'm sequencing 5Mb bacterial strains what type of coverage would I get from that one run (I know this is somewhat dependent on the MiSeq kit chosen)? Do people usually include less strains on a run or do multiple runs with many strains to ensure good coverage? Thanks!

[GenoMax]

You can usually get between 20-25 M reads (2 x 300 bp) from a MiSeq run. That translates to 12-15 gb of sequence data. You can determine theoretical coverage for "x" number of strains you choose to multiplex. Keep in mind that it is tricky to get uniform distribution for libraries when multiplexing. You may have to do test runs and re-pool/adjust if you are interested in a certain fold coverage per strain.

[Vanschkn]

Thanks for your reply. Do you have experience multiplexing bacterial strains? The Nextera kits advertise that they can multiplex up to 96 strains and the bead technology helps QC the libraries to ensure an even distribution. Any experience with this? I want to go in with realistic data expectations!

[GenoMax]

I will let someone else comment on the experimental part of your question.

As far as data goes, irrespective of the number of strains you choose to multiplex (we will go with an example of 5 strains) the total amount of data from one MiSeq run is going to be split 5 ways (12-15 gigabase total data, each strain would get 2.5-3 gb data).

Note: Your libraries have to be of reasonably good quality otherwise above would not apply.

[nucacidhunter]

Assuming using V3 with 2x300 cycle you will get around 20-25x coverage for 96 samples. If you need more coverage you can run your library pool multiple times to get required coverage. Though, if read length is not that important it might be more cost effective to run your library pool on HiSeq. With recent release of new primer set with Nextera, up to 384 plexing is possible. If you decide to run your libraries on HiSeq it would be better to stop after PCR clean-up and skip bead normalisation step.