Optimal coverage for RNA-seq analysis of DGE in bacteria

[seb330]

Hi all,

I am planning an experiment to look at DGE in a bacterial culture before and after nutrient depletion. We will be doing RNA-seq using an Illumina Hi-seq, and the facility has told us that they typically get 150-180 million reads/lane. I am wondering how many samples I can safely multiplex in one lane and still get enough depth to see differences in gene expression. I should have 4-6 time points in triplicate (12-18 samples). Any advice would be appreciated!

Thanks,
Sarah

[rflrob]

Hi Sarah,
I would feel pretty comfortable doing all of those in 1 lane. In our Drosophila lab (genome size ~ 170 MB, 17k genes), we typically multiplex 6-10 libraries per lane, so your bacteria should have plenty of coverage in just one.

[seb330]

Thanks, rflrob! That definitely makes me feel better about multiplexing these! Our genome is ~5Mb, so that would definitely be enough coverage based on your numbers. Are you able to see good, clear differences in gene expression with that many samples in a single lane?

Sarah

[rflrob]

Absolutely. Of course, the more power you can throw at it, the better you'll be able to distinguish subtle variations, but 3-5 million reads (per replicate with 3 replicates) is plenty to get clear differences in RPKMs as low as 10-20. Keep in mind, back in the good ol' days (i.e. a couple years ago, before HiSeqs), the GA IIx's would give you 5-20 million reads, and that was plenty for Eukaryotes.