[BioC] DiffBind error

[jluis.lavin at unavarra.es]

Dear list,

I made a new samplesheet to use with DiffBind with this format:

SampleID	Tissue	Factor	Condition	Replicate	bamReads	bamControl	Peaks
Contrl1	Neural	K9	Resistant	1	Control1.bed	Contro_input.bed	Control1_peaks.bed
Contrl2	Neural	K9	Resistant	2	Control2.bed	Control_input.bed	Control2_peaks.bed
A4_1	Neural	K9	Responsive	1	A4_1.bed	A4_input.bed	A4_1_peaks.bed
A4_2	Neural	K9	Responsive	2	A4_2.bed	A4_input.bed	A4_2_peaks.bed
A21_1	Neural	K9	Responsive	1	A21_1.bed	A21_input.bed	A21_1_peaks.bed
A21_2	Neural	K9	Responsive	2	A21_2.bed	A21_input.bed	A21_2_peaks.bed

I can load the files,

> chippy = dba(sampleSheet="Peaksets_sample_sheet.csv")

plot them

>plot(chippy)

and count the reads,

>chippy = dba.count(chippy, minOverlap=3)

but when I try to establish a contrast based on the condition metadata, I
get the following warning message:

> chippy = dba.contrast(chippy, categories=DBA_CONDITION)

Warning message:
No contrasts added. Perhaps try more categories, or lower value for
minMembers.

So when I try to perform the analysis, it doesn't work:

> chippy = dba.analyze(chippy)

Error in pv.DBA(DBA, method, bSubControl, bFullLibrarySize, bTagwise =
bTagwise,  :
  Unable to perform analysis: no contrasts specified.
In addition: Warning message:
No contrasts added. Perhaps try more categories, or lower value for
minMembers.

->My questions are:
-What is the contrast for DiffBind (I added the input for each set of
samples, and 2 biological replicates as control)?
-Is there something wrong with the sample sheet?
-Shouldn't the files to analyze be in bed format?

Thanks in advance

JL


El Jue, 20 de Diciembre de 2012, 14:36, jluis.lavin at unavarra.es escribió:
> Thank you Gordon, it was my mistake, I missunderstood the sample sheet and
> didn't create mine correctly.
> I think the packege is very very useful and I can't wait to see it working
> correctly with my samples  ;)
>
> Best wishes
>
> JL
>
>
> El Jue, 20 de Diciembre de 2012, 12:17, Gordon Brown escribió:
>> Hi,
>>
>> Your sample sheet shows the same filename for "Reads" and "Peaks", which
>> is unlikely to be right.  The "Peaks" file should be the output of the
>> peak caller (the peaks that MACS called); the "Reads" file should be the
>> aligned reads, i.e. the *input* to the peak caller (the output of the
>> alignment stage).  Let me know if that fixes the problem.
>>
>> It should give an informative error message, though... I'll add that to
>> the to-do list.
>>
>> Cheers,
>>
>>  - Gord
>>
>>
>>>----------------------------------------------------------------------
>>>
>>>Message: 1
>>>Date: Wed, 19 Dec 2012 16:17:07 +0100
>>>From: jluis.lavin at unavarra.es
>>>To: bioconductor at r-project.org
>>>Subject: [BioC] Diffbind error
>>>Message-ID:
>>>	<e6a0d8855033e937b5236f5787e1d78d.squirrel at webmail.unavarra.es>
>>>Content-Type: text/plain;charset=iso-8859-1
>>>
>>>Dear list,
>>>
>>>--I'm trying to use Diffbind package to analyze some samples from a ChIP
>>>seq experiment.
>>>--I created a .csv samplesheet with the following fields and which
>>> samples
>>>to analyze:
>>>
>>>SampleID Tissue	Factor	Condition Replicate Reads Control	Peaks
>>>
>>>Chip_K27.1+ Neural K27 Responsive 1 Chip_K27.BED Input_K.BED
>>> Chip_K27.BED
>>>
>>>Chip_K27.2+ Neural K27 Responsive 1 Chip_K27d.BED Input_K.BED
>>>Chip_K27d.BED
>>>
>>>--All my BED files have 6 lines and have been retrieved from MACS peak
>>>caller.
>>>
>>>--When Diffbind reads the sheet everything seems correct:
>>>
>>>> chippy = dba(sampleSheet="Peaksets_sample_sheet.csv")
>>>
>>>Chip_K27.1+ Neural K27 Responsive  1 raw
>>>Chip_K27.2+ Neural K27 Responsive  1 raw
>>>
>>>#it plots the correlation heatmap
>>>
>>>> plot(chippy)
>>>
>>>#But fails in this step after a while
>>>> chippy = dba.count(chippy, minOverlap=3)
>>>
>>>Error in read.table(fn, skip = skipnum) : no lines available in input
>>>
>>>#Alternatively if I try this command
>>>> chippy = dba.count(chippy)
>>>
>>>Error in cond$counts : $ operator is invalid for atomic vectors
>>>In addition: Warning message:
>>>In mclapply(arglist, fn, ..., mc.preschedule = TRUE, mc.allow.recursive
>>> =
>>>TRUE) :
>>>  all scheduled cores encountered errors in user code
>>>
>>>Would any of you shed any light on this issue?
>>>
>>>Best wishes and thanks in advance
>>>
>>>JL
>>>
>>>
>>>> sessionInfo()
>>>R version 2.15.1 (2012-06-22)
>>>Platform: x86_64-redhat-linux-gnu (64-bit)
>>>
>>>locale:
>>> [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
>>> [3] 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
>>> [7] LC_PAPER=C                 LC_NAME=C
>>> [9] LC_ADDRESS=C               LC_TELEPHONE=C
>>>[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
>>>
>>>attached base packages:
>>>[1] parallel  stats     graphics  grDevices utils     datasets  methods
>>>[8] base
>>>
>>>other attached packages:
>>>[1] DiffBind_1.4.0       Biobase_2.18.0       GenomicRanges_1.10.5
>>>[4] IRanges_1.16.4       BiocGenerics_0.4.0   BiocInstaller_1.8.3
>>>
>>>loaded via a namespace (and not attached):
>>> [1] amap_0.8-7         edgeR_3.0.7        gdata_2.12.0
>>>gplots_2.11.0
>>> [5] gtools_2.7.0       limma_3.14.3       RColorBrewer_1.0-5
>>>stats4_2.15.1
>>> [9] tools_2.15.1       zlibbioc_1.4.0
>>>
>>>
>>>--
>>>Dr. Jos? Luis Lav?n Trueba
>>>
>>>Dpto. de Producci?n Agraria
>>>Grupo de Gen?tica y Microbiolog?a
>>>Universidad P?blica de Navarra
>>>31006 Pamplona
>>>Navarra
>>>SPAIN
>>
>>
>
>
> --
> Dr. José Luis Lavín Trueba
>
> Dpto. de Producción Agraria
> Grupo de Genética y Microbiología
> Universidad Pública de Navarra
> 31006 Pamplona
> Navarra
> SPAIN
>
>
>


-- 
Dr. José Luis Lavín Trueba

Dpto. de Producción Agraria
Grupo de Genética y Microbiología
Universidad Pública de Navarra
31006 Pamplona
Navarra
SPAIN