To have access to RNA-seq data produced by MAQC project,
you'll have to be a member of MAQC Consortium. Have a look at the MAQC
website for details (http://www.fda.gov/ScienceResearch/Bioinformatics
The TaqMan RT-PCR data is publicly available, which can be
downloaded from GEO (GSE5350).
On Apr 29, 2011, at 5:41 PM, Stefano Calza wrote:
> Dr Wei
> are these data on RNA-seq and RT-PCR already available?
> On Fri, Apr 29, 2011 at 01:26:49PM +1000, Wei Shi wrote:
> <wei>Hi Fernando:
> <wei> We had some positive control genes which we know should be up
-/down-regulated in one cell type compared to the other from previous
RT-PCR experiments. The quantile method successfully detected all
these control genes and gave them higher ranks in the list of
differentially expressed genes compared to other normalization
methods. You could certainly argue that this is a biased comparison,
but when you do not know which method works best, the one which gives
results more closer to your expectation is often preferred.
> <wei> My belief in the quantile method actually mainly came from a
evaluation study using the RNA-seq data from MAQC project, in which
expression levels of ~1000 genes were validated by RT-PCR. What I
found was that the quantile normalized data had a better correlation
with the PCR data, compared to other normalization methods. This work
hasn't been published yet, but I am working on that.
> <wei>On Apr 29, 2011, at 12:51 PM, Biase, Fernando wrote:
> <wei>> Dr. Wei,
> <wei>> If I may I ask. What criteria do you use to find out which
normalization suits better your data?
> <wei>> thanks,
> <wei>> Fernando
> <wei>> ________________________________________
> <wei>> From: bioconductor-bounces at r-project.org [bioconductor-
bounces at r-project.org] On Behalf Of Wei Shi [shi at wehi.EDU.AU]
> <wei>> Sent: Thursday, April 28, 2011 6:07 PM
> <wei>> To: Jo??o Moura
> <wei>> Cc: bioconductor at r-project.org list
> <wei>> Subject: Re: [BioC] RNASeq: normalization issues
> <wei>> Hi Jo??o:
> <wei>> Maybe you can try different normalization methods for
your data to see which one looks better. How to best normalize RNA-seq
data is still of much debate at this stage.
> <wei>> You can try scaling methods like TMM, RPKM, or 75th
percentile, which as you said normalize data within samples. Or you
can try quantile between-sample normalization (read counts should be
adjusted by gene length first), which performs normalization across
samples. You can try all these in edgeR package.
> <wei>> From my experience, I actually found the quantile
method performed better for my RNA-seq data. I used general linear
model and likelihood ratio test in edgeR in my analysis.
> <wei>> Hope this helps.
> <wei>> Cheers,
> <wei>> Wei
> <wei>> On Apr 28, 2011, at 7:36 PM, Jo??o Moura wrote:
> <wei>>> Dear all,
> <wei>>> Until now I was doing RNAseq DE analysis and to do that I
> <wei>>> normalization issues only matter inside samples, because one
can assume the
> <wei>>> length/content biases will cancel out when comparing same
genes in different
> <wei>>> samples.
> <wei>>> Although, I'm now trying to compare correlation of different
genes and so,
> <wei>>> this biases should be taken into account - for this is there
> <wei>>> method than RPKM?
> <wei>>> My main doubt is if I should also take into acount the
biases inside samples
> <wei>>> and to do that is there any better approach then TMM by
Robinson and Oshlack
> <wei>>> ?
> <wei>>> Thank you all,
> <wei>>> --
> <wei>>> Jo??o Moura
> <wei>>> [[alternative HTML version deleted]]
> <wei>>> _______________________________________________
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> <wei>>> Search the archives:
> <wei>> The information in this email is confidential and
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> <wei>Search the archives:
> Stefano Calza, PhD
> Researcher/Assistent Professor - Biostatistician
> *Sezione di Statistica Medica e Biometria
> Dipartimento di Scienze Biomediche e Biotecnologie
> Universit? degli Studi di Brescia - Italy
> Viale Europa, 11 25123 Brescia
> email: stefano.calza at med.unibs.it
> stefano.calza at biostatistics.it
> pec: stefano.calza at pec.biostatistics.it
> Phone: +390303717653
> Fax: +390303717488
The information in this email is confidential and