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Mark Robinson
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@mark-robinson-4908
Last seen 6.1 years ago
Dear Sooby,
Please keep posts on the list and see:
http://bioconductor.org/help/mailing-list/posting-guide/
On 21.04.2012, at 18:17, Sooby wrote:
> I'm not sure about the library size you mentioned.
> Is it the total reads of a sample or the sum of the reads mapped to
each gene.
> I mean, if the total reads of a sample is 1,000,100,
> but when I mapped the reads to the gene, I got a list of gene with
reads mapped to it, and the sum of this is 900,000.
> Do I use 1,000,000 or 900,000??
I feel like I answered this already. My previous response was
"Usually, total number of mapped reads.", which would suggest 900,000
in your example.
> The normalization factor I calculate is like 0.5,1.5,1.3 ,but in you
manual they are all near to 1.
> Is it right??
It is more extreme than I normally see, but it could be ok. Have you
looked at your data? Figure 1 of the edgeR user's guide gives an
example "smear" plot. Perhaps you should look at one of those for
your data and assess whether the normalization factors are
appropriate.
Best,
Mark
> ???? Mark Robinson <mark.robinson at="" imls.uzh.ch="">
> ???? Sooby <sooby24 at="" yahoo.cn="">
> ??? "bioconductor at r-project.org" <bioconductor at="" r-project.org="">
> ????? 2012?4?21?, ???, ?? 5:20
> ??: Re: [BioC] TMM factors
>
> Hi Sooby,
>
> Some comments below.
>
> On 20.04.2012, at 04:32, Sooby wrote:
>
> > Hi all,
> > I met a question when I analysed mRNA data. I use TMM provided in
edgeR to normalizition my data. After I got the factors, How Do I
treat my raw counts?? In my oppion,
(raw.counts)*10^7/(librarysize*factors),Is It right?
>
> So, this is correct, for a reads per 10M conversion. I'm not sure
what you mean by "treat my raw counts". If you are doing differential
expression with edgeR (or DESeq, etc.), we strongly suggest to use the
raw counts, without "treating" them.
>
>
> > And another question is which librarysize do I need? The unique
total reads or the reads mapped to each gene???
>
> Usually, total number of mapped reads.
>
> Best,
> Mark
>
> > I need your help~~~
> > Best
> >
> > [[alternative HTML version deleted]]
> >
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