Hello Mark,
If I want to include a term (Gene length) in the below mentioned code to
make it like RPKM. How to add this term. I would appreciate it.

Many thanks in advance!

Sridhara


On Wed, Feb 9, 2011 at 6:19 PM, Sridhara Gupta Kunjeti <sridhara@udel.edu>wrote:

> Hello Mark,
> Yes, Now it is clear to me.
> Thank you very much for being patient in responding to my questions.
>
> Many thanks!
> Sridhara
>
>
> On Wed, Feb 9, 2011 at 5:16 PM, Mark Robinson <mrobinson@wehi.edu.au>wrote:
>
>> Hi Sridhara.
>>
>> On 2011-02-10, at 4:34 AM, Sridhara Gupta Kunjeti wrote:
>>
>> > Hello Mark,
>> > This is in continuation with the normalization of the counts:
>> > did you mean
>> >
>> > (count / library size) * Norm.factor
>> > Can I use the numbers for the library size and Norm.factor can be used
>> from the edgeR?
>>
>>
>> No.  Actually, I mean what I wrote in both previous posts.  I'll repeat
>> again and hopefully third time lucky:
>>
>> rpm <- t(t(d$counts) / (d$samples$lib.size*d$samples$norm.factors)) * 1e6
>>
>> So, this translates to:
>>
>> count / (lib.size*Norm.factor)
>>
>> ... and you may multiply by a factor to put it on a different scale (e.g.
>> multiply by 1M as I've done above).  And, you should remember all the
>> previous caveats that I've mentioned (i.e. there is no need to do this for a
>> differential expression analysis as edgeR already builds this in + this
>> doesn't account for other biases such as gene length).
>>
>> Hope that helps.
>> Mark
>>
>>
>>
>>
>> > Thanks,
>> > Sridhara
>> >
>> >
>> > On Mon, Feb 7, 2011 at 5:11 PM, Mark Robinson <mrobinson@wehi.edu.au>
>> wrote:
>> > Hi Jens/Sridhara.
>> >
>> > A few thoughts below.
>> >
>> > On 2011-02-07, at 11:22 PM, Sridhara Gupta Kunjeti wrote:
>> >
>> > > Hi Gordon,
>> > > First I would like to thank Jens for asking the questions that I had
>> asked
>> > > few days ago.
>> > > In additions to the Jens question, I have one more question on my
>> RNA-seq
>> > > data
>> > > 1. I would like to know if I can multiply the counts for each gene
>> with the
>> > > norm.factor (calculated by "calcNormFactors( )" function)
>> >
>> >
>> > Sridhara, you've asked this exact question before and I answered (short
>> answer is: NO to multiplying ... instead, divide by [library
>> size]*[normalization factor]):
>> >
>> > https://stat.ethz.ch/pipermail/bioconductor/2011-January/037564.html
>> > https://stat.ethz.ch/pipermail/bioconductor/2011-January/037469.html
>> >
>> > Perhaps you can clarify what you don't understand.
>> >
>> >
>> > > On Mon, Feb 7, 2011 at 5:46 AM, Jens Georg <
>> > > jens.georg@biologie.uni-freiburg.de> wrote:
>> > >
>> > >> Hi Gordon,
>> > >> thank you for your reply. The resolution of our ~100nt solexa reads
>> is to
>> > >> small to detect individual processing sites, so we want to
>> investigate every
>> > >> single nucleotide individually ("single nucleotide based
>> normalization").
>> > >> That means that we count, how often an individual nucleotide is
>> covered by
>> > >> sequence reads. Of course, this approach will virtually increase the
>> > >> lib.size by a factor which depends on length of the solexa reads. As
>> the
>> > >> lib.size is critical for the normalization, I am not sure if I should
>> use
>> > >> the original read numbers for each library or the read numbers
>> multiplicated
>> > >> with the read length to adjust for the single nucleotide
>> investigation.
>> >
>> >
>> > So basically, by counting this way, your library size is ~100x the
>> number of reads you've actually mapped.  While I think this will work out ok
>> (normalization calculation be fine), this coverage calculation does impose a
>> (strong?) dependence between adjacent nucleotides.  One alternative would be
>> to count the reads that *begin* at a given nucleotide and only consider
>> these.  Then your library sizes are as normal.
>> >
>> >
>> > >> I have two more question regarding to the normalization:
>> > >> 1. Are the norm factors calculated by the calcNormFactors( ) function
>> > >> automatically used for further steps like the estimateCommonDisp( )
>> > >> function?
>> >
>> > Yes.
>> >
>> >
>> > >> 2. Are the pseudocounts calculated by estimateCommonDisp( ) the
>> normalized
>> > >> readcounts?
>> >
>> > Yes, but this is only accounting for overall depth and potential
>> composition biases, not for length biases (or any others).  It is with the
>> intention of making inferences of a given gene across conditions.  The
>> inferences for differential expression are still done on the raw counts.
>> >
>> > Hope that helps.
>> > Mark
>> >
>> >
>> >
>> >
>> > >>
>> > >> Many thanks
>> > >>
>> > >> Jens
>> > >>
>> > >> Hi Jens,
>> > >>>
>> > >>> I don't know what you mean by single nucleotide based normalization,
>> > >>> however the following comments may be helpful.
>> > >>>
>> > >>> edgeR automatically adjusts for library sizes, whether you include
>> an
>> > >>> explicit normalization step or not.  Normalization is a separate
>> issue, and
>> > >>> is intended to deal with more subtle issues.
>> > >>>
>> > >>> Normalization, as edgeR does it, does not require replicates.
>> > >>>
>> > >>> Best wishes
>> > >>> Gordon
>> > >>>
>> > >>> Date: Fri, 04 Feb 2011 11:28:15 +0100
>> > >>>> From: Jens Georg <jens.georg@biologie.uni-freiburg.de>
>> > >>>> To: bioconductor@r-project.org
>> > >>>> Subject: [BioC] Single nucleotide based RNAseq normalization with
>> > >>>>   edgeR?
>> > >>>> Message-ID: <4D4BD4BF.4010009@biologie.uni-freiburg.de>
>> > >>>> Content-Type: text/plain; charset=ISO-8859-15; format=flowed
>> > >>>>
>> > >>>>
>> > >>>>
>> > >>>> Dear edgeR users and developers,
>> > >>>>
>> > >>>> we used Solexa sequencing in order to detect RNase E processing
>> sites.
>> > >>>> Therefor we splitted a RNA sample and treated one half with RNase E
>> > >>>> prior to cDNA synthesis and sequencing. The libraries differ in
>> size
>> > >>>> (1.918.953 and 1.208.586 reads respectively) which clearly
>> necessitates
>> > >>>> a normalization step. Furthermore we expect site specific
>> differences
>> > >>>> rather than differences in the accumulation of the full length
>> RNAs.
>> > >>>>
>> > >>>> So I want to ask, if it is appropiate to do a single nucleotide
>> based
>> > >>>> normalization with edgeR and do you think a reliable basic
>> normalization
>> > >>>> is possible without replicates?
>> > >>>>
>> > >>>> Thank you for your comments.
>> > >>>>
>> > >>>> Best regards
>> > >>>>
>> > >>>> Jens
>> > >>>>
>> > >>>
>> > >>>
>> ______________________________________________________________________
>> > >>> The information in this email is confidential and
>> inte...{{dropped:6}}
>> > >>>
>> > >>
>> > >> _______________________________________________
>> > >> Bioconductor mailing list
>> > >> Bioconductor@r-project.org
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>> > >> Search the archives:
>> > >> http://news.gmane.org/gmane.science.biology.informatics.conductor
>> > >>
>> > >
>> > >
>> > >
>> > > --
>> > > Sridhara G Kunjeti
>> > > PhD Candidate
>> > > University of Delaware
>> > > Department of Plant and Soil Science
>> > > email- sridhara@udel.edu
>> > > Ph: 832-566-0011
>> > >
>> > >       [[alternative HTML version deleted]]
>> > >
>> > > _______________________________________________
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>> > > https://stat.ethz.ch/mailman/listinfo/bioconductor
>> > > Search the archives:
>> http://news.gmane.org/gmane.science.biology.informatics.conductor
>> >
>> > ------------------------------
>> > Mark Robinson, PhD (Melb)
>> > Epigenetics Laboratory, Garvan
>> > Bioinformatics Division, WEHI
>> > e: mrobinson@wehi.edu.au
>> > e: m.robinson@garvan.org.au
>> > p: +61 (0)3 9345 2628
>> > f: +61 (0)3 9347 0852
>> > ------------------------------
>> >
>> >
>> > ______________________________________________________________________
>> > The information in this email is confidential and intended solely for
>> the addressee.
>> > You must not disclose, forward, print or use it without the permission
>> of the sender.
>> > ______________________________________________________________________
>> >
>> >
>> >
>> > --
>> > Sridhara G Kunjeti
>> > PhD Candidate
>> > University of Delaware
>> > Department of Plant and Soil Science
>> > email- sridhara@udel.edu
>> > Ph: 832-566-0011
>>
>> ------------------------------
>> Mark Robinson, PhD (Melb)
>> Epigenetics Laboratory, Garvan
>> Bioinformatics Division, WEHI
>> e: mrobinson@wehi.edu.au
>> e: m.robinson@garvan.org.au
>> p: +61 (0)3 9345 2628
>> f: +61 (0)3 9347 0852
>> ------------------------------
>>
>>
>> ______________________________________________________________________
>> The information in this email is confidential and intended solely for the
>> addressee.
>> You must not disclose, forward, print or use it without the permission of
>> the sender.
>> ______________________________________________________________________
>>
>
>
>
> --
> Sridhara G Kunjeti
> PhD Candidate
> University of Delaware
> Department of Plant and Soil Science
> email- sridhara@udel.edu
> Ph: 832-566-0011
>



-- 
Sridhara G Kunjeti
PhD Candidate
University of Delaware
Department of Plant and Soil Science
email- sridhara@udel.edu
Ph: 832-566-0011

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