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Old 12-17-2013, 02:33 AM   #1
Synthetica
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Location: London

Join Date: Dec 2013
Posts: 4
Default 96-sample RNAseq: nextera, hiseq, ribo-zero

Hi,

My lab are new to next-gen sequencing and we're planning an RNAseq experiment with a bunch of E. coli samples and different time-points. We'd like to max-out 1 lane on a HiSeq with 96 different samples. Coverage depth may not be that good, but that's something we can deal with as our experiment is designed simply to identify the promoters that up- and down-regulate their expression the most during the time-points, hence we are not that interested in small RNAs, regulatory RNAs etc. Just good old-fashioned transcripts.

From what I can tell, Nextera barcoding will get us access to 96 samples in one lane of a HiSeq and the facility we will use in London estimates that the E.coli transcriptome done 96 times will fit on one lane of their HiSeq with enough depth for what we are after. Is this true? I have doubts as the facility have never done a bacterial RNAseq before.

The next question is whether we need to remove ribosomal RNA. In bacteria the amount of RNA that is rRNA is debated but estimates range from 80 to 98%. I'm inclined to believe it is 85% from a fancy paper in Science in 2010. Removal of this could therefore increase our read depth by about 7-fold. Right? However, for the same price as the four kits we need to buy from Epicenter to process 96 samples it looks like we could simply buy-up another 4 or 5 lanes on the HiSeq and increase the depth of read that way. Which is the better option?

The Epicenter kits are damn expensive in the UK and they don't do a 96 sample one. I hope I'm not missing a trick here - is there a way to barcode samples first, then pool and remove rRNA in one lot just before sequencing? Then we'd only need 1 kit.

I hope some of this awesome community can help us with some of our questions. I'll buy a beer for you if you're in London.

Cheers
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Old 12-17-2013, 08:27 AM   #2
oligo
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Location: Europe

Join Date: Dec 2011
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Default More like 95%-98%

In bacterial RNA-seq libs without depletion you will get 95-98% rRNA. Depletion is almost essential and there are several methods to achieve that (ribo-zero, microbexpress, homebrew RNaseH).
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e.coli, hiseq, nextera xt, ribo-zero, rnaseq library prep

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