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Old 06-04-2013, 09:26 PM   #2
Macrophage
Junior Member
 
Location: Quebec City

Join Date: Jun 2013
Posts: 4
Question idem

Hi Frazzled! This is probably too late but I would like the discussion to be open again. 1 year after you, I have exactly the same question and have some troubles to find someone who has already used it for RNAseq. Everybody around me tells, this is absolutely needed to compare results from different platforms but nobody use it or at least knows someone that would have been eared about a guy who would have already used it.
This approach is strongly recommended by the ENCODE consortium (http://genome.ucsc.edu/ENCODE/protoc...dards_V1.0.pdf + https://www.google.ca/url?sa=t&rct=j...47380653,d.aWM + http://nxseq.bitesizebio.com/article...-its-solution/).

However, I could only find 3 papers seriously considering this and performing some pilot experiments (http://genome.cshlp.org/content/21/9/1543.long + http://www.sciencedirect.com/science...92867412012263 + http://link.springer.com/article/10....427-013-4437-9)

I contacted both Illumina and Epicentre and they don't really care about this. Epicentre just told me that one customer lost half of reads in spike-in sequencing (I guess he/she should have put a huge amount, I have no information on it).
"[...] we do not normally recommend them, and I just spoke with a customer overseas who did use them and it completely "swamped out" the data from the sample such that the spiked RNA provided the bulk of the data. Remember that ScriptSeq uses a very minimal amount of RNA (500 pg to 50 ng) and we'd be worried about how much of the data generated would be from the bona fide sample versus the spiked in RNA."

Staff Technical Applications Scientist
Epicentre (an Illumina company)
(800) 284-8474, ext 66119
608.442.6119
Website: www.epicentre.com

My conclusion is that one who wants to use it should perform a pilot experiment (what should actually be done for any RNAseq analysis). I know because of time and money, this is not always possible. In my case, I have neither time nor money for pilot experiments so I'll go (with deep regrets) to library construction without spike-in controls and assess like many others that the coverage and global expression levels are not significantly variable between my samples (what I guess is not true).
Anyway, there is no perfect experiment and there are probably many bad things we are doing and we don't now yet. For sure, RNAseq standards are not fixed forever.

I would be glad to ear different opinions if somebody already included these controls.

Last edited by Macrophage; 06-04-2013 at 09:29 PM.
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