[BioC] Experimental design for RNA-Seq

[Steve Lianoglou]

Hi,

On Fri, May 28, 2010 at 12:04 PM, michael watson (IAH-C)
<michael.watson at bbsrc.ac.uk> wrote:
> Great stuff, thanks Steve and Naomi.
>
> I guess I was thinking of technical replicates simply as sequencing the same library on multiple occasions;  though creating two libraries out of one sample adds an extra layer of complexity.
>
> What is the evidence (if any) that lane and/or library preparation can have an effect?

I'm sure that this has hit your radar already, but I should point out
that at least some part of the recent Bullard et al. paper:
http://www.biomedcentral.com/1471-2105/11/94

talks about lane, flow-cell, and library prep effects. In their
concluding remarks, the mention that:

"""We find that technical variation is quite low across lanes and
flow-cells and slightly larger across library preparations. In all
cases, however, the effect on differ- ential expression results is
minimal. As noted above, the MAQC datasets are unusual, in that we
expect extre- mely large differences in expression between Brain and
UHR and only small library preparation effects because of the high
quality of the RNA. In practice, library pre- paration effects may be
closer in magnitude to biological effects."""

I feel like their original "working draft" paper that you can get from bepress:
http://www.bepress.com/ucbbiostat/paper247

talks about lane effects too (if the BMC Bioinformatics one doesn't)
.. I can't recall .. it might be worth looking at, too.

I'm also reminded of some comparisons that were performed in the
"FRT-seq" paper:

http://www.nature.com/nmeth/journal/v7/n2/full/nmeth.1417.html

They were presenting a modified Illumina sequencing protocol that
skips the traditional amplification step, and prepare two libraries
from the same sample using their FRT-seq protocol, and the "standard"
Illumina rna-seq protocol, to show you the pros/cons of each.

There is data is also made available for you to play with, if you're
so inclined.

If you have any vote on how the sequencing is done, though, and you
guys are using an Illumina sequencer, it seems like doing FRT-seq
would be a big win (if you were to ask me :-)

-steve
-- 
Steve Lianoglou
Graduate Student: Computational Systems Biology
 | Memorial Sloan-Kettering Cancer Center
 | Weill Medical College of Cornell University
Contact Info: http://cbio.mskcc.org/~lianos/contact