Entering edit mode
Dror Hibsh
▴
40
@dror-hibsh-5343
Last seen 10.1 years ago
Maybe it is better for 3 conditions to use GLM?
On Mon, Jun 18, 2012 at 6:17 PM, Dror Hibsh <drordh@gmail.com> wrote:
> Thanks Simon,
> Can you please recommend me on different tool for quantification
that
> suitable with DESeq?
>
> Thanks,
> Pap
>
>
> On Mon, Jun 18, 2012 at 10:20 AM, Dror Hibsh <drordh@gmail.com>
wrote:
>
>> i am doing de novo assembly, that is why i am using raw
reads(unaligned)
>> Thanks!
>>
>>
>> On Mon, Jun 18, 2012 at 8:24 AM, Dror Hibsh <drordh@gmail.com>
wrote:
>>
>>> Hi Tim, Thanks for the response!
>>> No i didnt..
>>> Is there any recommended tool for removing the pcr duplicates from
raw
>>> reads?
>>>
>>> Thanks,
>>> Pap
>>>
>>>
>>>
>>> On Sun, Jun 17, 2012 at 7:29 PM, Tim Triche, Jr.
<tim.triche@gmail.com>wrote:
>>>
>>>> you say you are "new to this field" yet you seem to have done
almost
>>>> everything right.
>>>>
>>>> have you tried removing PCR dupes that might be skewing your
results,
>>>> before transcript assembly with RSEM and DE testing?
>>>>
>>>>
>>>>
>>>> On Sun, Jun 17, 2012 at 9:23 AM, papori [guest]
<guest@bioconductor.org>>>> > wrote:
>>>>
>>>>>
>>>>> Hi all,
>>>>> First of all, I am new to this field so i am sorry if i am not
clear..
>>>>>
>>>>> I will try to explain what is my aim, and what i did before
DESeq.
>>>>>
>>>>> I am trying to do Differential expression analysis using DESeq
for
>>>>> De-Novo invertebrate .
>>>>>
>>>>> We had an experiment of 3 conditions with 3 biological replicate
for
>>>>> each.(total of 9 samples)
>>>>> We used hiseq2000 50bp single end reads.
>>>>> We had a different library size for each.(that was single cell
>>>>> experiment so we had amplification step.. what yield variance in
the
>>>>> library sizes..)
>>>>>
>>>>> We reconstructed the transcriptome using Trinity.
>>>>> Estimating counts with RSEM.
>>>>>
>>>>> And then i used DESeq..
>>>>>
>>>>> i have weird behavior of the data, and i dont know if it is
because
>>>>> something wrong that i did..
>>>>>
>>>>> i am always getting down-expression from condition 1 to
condition 2
>>>>> and high-expression from condition 2 to condition 3.(for all the
>>>>> transcripts, no out-layers..)
>>>>>
>>>>> The number of counts that got for each condition to reference
>>>>> transcriptome was:
>>>>> 32M, 27M, 40M respectively..
>>>>> What made me to think that because cond 2 has lowest count it
has a
>>>>> behavior of down-expression from 1 to 2 and high-expression from
2 to 3..
>>>>>
>>>>> if my conclusion is right, i am in a big mass..(Normalization??)
>>>>>
>>>>> my DESeq script is:
>>>>> Conditions = c("C1", "C2", "C3", "C1", "C2", "C3","C1", "C2",
"C3")
>>>>> Counts<-round(MultiGeneMat,0)
>>>>> cds <- newCountDataSet(Counts,Conditions)
>>>>> cds <- estimateSizeFactors(cds)
>>>>> cds <-
>>>>> estimateDispersions(cds,method="per-
condition",sharingMode="maximum",fitType="local")
>>>>>
>>>>> res_1vs2 <- nbinomTest(cds,condA="C1",condB="C2")
>>>>> sigDESeq_1vs2 <- res_1vs2[res_1vs2$padj <= 0.1, ]
>>>>> sigDESeq_1vs2 <- na.omit(sigDESeq_1vs2)
>>>>>
>>>>> res_2vs3 <- nbinomTest(cds,condA="C2",condB="C3")
>>>>> sigDESeq_2vs3 <- res_2vs3[res_2vs3$padj <= 0.1, ]
>>>>> sigDESeq_2vs3 <- na.omit(sigDESeq_2vs3)
>>>>>
>>>>> res_1vs3 <- nbinomTest(cds,condA="1",condB="C3")
>>>>> sigDESeq_1vs3 <- res_1vs3[res_1vs3$padj <= 0.1, ]
>>>>> sigDESeq_1vs3 <- na.omit(sigDESeq_1vs3)
>>>>>
>>>>>
>>>>>
>>>>> Is there anything wrong here? or anywhere else??
>>>>> If i wasnt clear enough so tell me in what and i will try to
explain..
>>>>> Any help will be appreciate here!
>>>>> Thanks,
>>>>> Pap
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> -- output of sessionInfo():
>>>>>
>>>>> R version 2.14.0 (2011-10-31)
>>>>> Platform: x86_64-unknown-linux-gnu (64-bit)
>>>>>
>>>>> locale:
>>>>> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
>>>>> LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
>>>>> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
LC_PAPER=C
>>>>> LC_NAME=C
>>>>> [9] LC_ADDRESS=C LC_TELEPHONE=C
>>>>> LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
>>>>>
>>>>> attached base packages:
>>>>> [1] stats graphics grDevices utils datasets methods
base
>>>>>
>>>>> other attached packages:
>>>>> [1] edgeR_2.4.6 limma_3.10.3 DESeq_1.6.1 locfit_1.5-8
>>>>> Biobase_2.14.0
>>>>>
>>>>> loaded via a namespace (and not attached):
>>>>> [1] annotate_1.32.3 AnnotationDbi_1.16.19 DBI_0.2-5
>>>>> genefilter_1.36.0 geneplotter_1.32.1
>>>>> [6] grid_2.14.0 IRanges_1.12.6 lattice_0.20-6
>>>>> RColorBrewer_1.0-5 RSQLite_0.11.1
>>>>> [11] splines_2.14.0 survival_2.36-14 tools_2.14.0
>>>>> xtable_1.7-0
>>>>> >
>>>>>
>>>>>
>>>>> --
>>>>> Sent via the guest posting facility at bioconductor.org.
>>>>>
>>>>> _______________________________________________
>>>>> Bioconductor mailing list
>>>>> Bioconductor@r-project.org
>>>>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>>>>> Search the archives:
>>>>>
http://news.gmane.org/gmane.science.biology.informatics.conductor
>>>>>
>>>>
>>>>
>>>>
>>>> --
>>>> *A model is a lie that helps you see the truth.*
>>>> *
>>>> *
>>>> Howard
Skipper<http: cancerres.aacrjournals.org="" content="" 31="" 9="" 1173.full.pdf="">
>>>>
>>>>
>>>
>>>
>>> --
>>> -----------------
>>> Dror Hibsh
>>> 0507-669599
>>> ------------------
>>>
>>>
>>
>>
>> --
>> -----------------
>> Dror Hibsh
>> 0507-669599
>> ------------------
>>
>>
>
>
> --
> -----------------
> Dror Hibsh
> 0507-669599
> ------------------
>
>
--
-----------------
Dror Hibsh
0507-669599
------------------
[[alternative HTML version deleted]]