HI Dr. Smyth,
I am trying to find the appropriate mRNA expression level estimates to
use for generating plots that demonstrate the differential expression
statistics calculated from voom/limma. What do you recommend? FPKM
plots don't always agree with my voom/limma results.
For example, if the differential expression results from limma report
a log Fold change of 2 for group A vs. group B, I want a plot (e.g.
boxplot, bar graph, etc.) which visualizes this difference and also
provides a realistic estimate of the expression level along the axis.
If you have other types of visualization (or normalizations) that you
prefer for showing estimated expression levels between groups that
also demonstrate the limma differential expression trends, please
share. I'm open to any ideas/suggestions that you have.
Thanks,
Heather
[[alternative HTML version deleted]]
Dear Heather,
voom() outputs normalized log-cpm values. Just plot them.
For example after
v <- voom(y, design)
the log-cpm values are in v$E.
Best wishes
Gordon
> Date: Thu, 6 Feb 2014 15:27:59 -0800
> From: Heather Estrella <hestrella at="" regulusrx.com="">
> To: "bioconductor at r-project.org" <bioconductor at="" r-project.org="">
> Subject: [BioC] What RNA-Seq expression estimates to use for
plotting
> to show voom/limma Differential expression
>
> HI Dr. Smyth,
>
> I am trying to find the appropriate mRNA expression level estimates
to
> use for generating plots that demonstrate the differential
expression
> statistics calculated from voom/limma. What do you recommend? FPKM
plots
> don't always agree with my voom/limma results.
>
> For example, if the differential expression results from limma
report a
> log Fold change of 2 for group A vs. group B, I want a plot (e.g.
> boxplot, bar graph, etc.) which visualizes this difference and also
> provides a realistic estimate of the expression level along the
axis.
>
> If you have other types of visualization (or normalizations) that
you
> prefer for showing estimated expression levels between groups that
also
> demonstrate the limma differential expression trends, please share.
I'm
> open to any ideas/suggestions that you have.
>
> Thanks,
> Heather
______________________________________________________________________
The information in this email is confidential and
intend...{{dropped:4}}
Please keep the discussion on the Bioconductor mailing list.
On Sat, 8 Feb 2014, Heather Estrella wrote:
> Thank you for responding. The CPM in the voom structure is what I
have
> been using. My concern is that I am not confident I can use CPM to
> compare across genes.
You can't use CPM to compare across genes, but you can use it to
display
differential expression between conditions, which is what I understood
you
to be asking for.
> The key set of questions I am trying to answer are
> the following:
>
> 1. What is the estimated expression level of gene A and how does
that level compare to a predefined set of other genes?
Just take the log2CPM values from voom, and subtract
log2(GeneLength/1000)
from each row, where GeneLength is the effective length of each gene
in
bases. This will convert the log2CPM values into log2 FPKM.
Best wishes
Gordon
> 2. Is gene A differentially expressed between 2 sample groups?
>
> I can visually represent this in a couple of boxplots or bargraphs,
one
> for each gene, as long as the estimated expression levels are
comparable
> and realistic.
>
> Thanks,
> Heather
>
>
>
>
> -------- Original message --------
> From: Gordon K Smyth <smyth at="" wehi.edu.au="">
> Date: 02/07/2014 11:28 PM (GMT-08:00)
> To: Heather Estrella <hestrella at="" regulusrx.com="">
> Cc: Bioconductor mailing list <bioconductor at="" r-project.org="">
> Subject: What RNA-Seq expression estimates to use for plotting to
show voom/limma Differential expression
>
>
> Dear Heather,
>
> voom() outputs normalized log-cpm values. Just plot them.
>
> For example after
>
> v <- voom(y, design)
>
> the log-cpm values are in v$E.
>
> Best wishes
> Gordon
>
>> Date: Thu, 6 Feb 2014 15:27:59 -0800
>> From: Heather Estrella <hestrella at="" regulusrx.com="">
>> To: "bioconductor at r-project.org" <bioconductor at="" r-project.org="">
>> Subject: [BioC] What RNA-Seq expression estimates to use for
plotting
>> to show voom/limma Differential expression
>>
>> HI Dr. Smyth,
>>
>> I am trying to find the appropriate mRNA expression level estimates
to
>> use for generating plots that demonstrate the differential
expression
>> statistics calculated from voom/limma. What do you recommend? FPKM
plots
>> don't always agree with my voom/limma results.
>>
>> For example, if the differential expression results from limma
report a
>> log Fold change of 2 for group A vs. group B, I want a plot (e.g.
>> boxplot, bar graph, etc.) which visualizes this difference and also
>> provides a realistic estimate of the expression level along the
axis.
>>
>> If you have other types of visualization (or normalizations) that
you
>> prefer for showing estimated expression levels between groups that
also
>> demonstrate the limma differential expression trends, please share.
I'm
>> open to any ideas/suggestions that you have.
>>
>> Thanks,
>> Heather
______________________________________________________________________
The information in this email is confidential and
intend...{{dropped:4}}
