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 escribi?: > 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 escribi?: >> 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. 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. 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

Hi, By default, dba.contrast won't create contrasts with less than 3 members in each group. The easiest solution is to set minMembers=2 when calling dba.contrast: > chippy = dba.contrast(chippy, categories=DBA_CONDITION, minMembers=2) Or you can create the contrast explicitly: > chippy = dba.contrast(chippy, group1=chippy$masks$Resistant, >group2=chippy$masks$Responsive, name1="a name", name2="another name") Hope this helps... Cheers, - Gord On 2013-01-29 14:21, "jluis.lavin at unavarra.es" <jluis.lavin at="" unavarra.es=""> wrote: >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_peak >s.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 escribi?: >> 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 escribi?: >>> 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. 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. 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 > >

Hello- It looks like your sample sheet is fine. By default, if no contrasts are set up using dba.contrast, when you call dba.analyze it attempts to add all the two-way contrasts between groups where there are at least three samples in each group. In your case, nothing meets these conditions, as there are only two "Resistant" samples. If you add a call to dba.contrast before the call to dba.analyze, you will get the contrast you desire: > chippy = dba.contrast(chippy, minMembers=2) or, slightly more explicitly: > chippy = dba.contrast(chippy, categories=DBA_CONDITION, minMembers=2) or, even more explicitly: > chippy = dba.contrast(chippy, group1=chippy$masks$Resistant, group2 = >chippy$masksResponsive, name1="Resistant", name2="Responsive") then: > chippy = dba.analyze(chippy) Cheers- Rory On Tue, 29 Jan 2013 15:21:23 +0100, jluis.lavin at unavarra.es wrote: > >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_peak >s.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 > >We may monitor all incoming and outgoing emails in line with current >legislation. We have taken steps to ensure that this email and >attachments are free from any virus, but it remains your responsibility >to ensure that viruses do not adversely affect you. >Cancer Research UKRegistered charity in England and Wales (1089464), >Scotland (SC041666) and the Isle of Man (1103) >A company limited by guarantee. Registered company in England and Wales >(4325234) and the Isle of Man (5713F). >Registered Office Address: Angel Building, 407 St John Street, London >EC1V 4AD.

Hello again, Thank you very much to Rory and Gordon for your kind and accurate help. Changing the minMembers parameter everything seemed to work fine and I've been able to perform the next steps of the whole analysis. Now I'm struggling with the retrieval of the analysis data; when I try to write the results into a .csv table with a simple command I get the following error: > write.csv( chippy, file="ChIPseq_diffbind.csv" ) Error in as.data.frame.default(x[[i]], optional = TRUE, stringsAsFactors = stringsAsFactors) : cannot coerce class '"DBA"' into a data.frame I read DiffBind's vignette and manual and I found the "save" command, but it doesn't allow me to retrieve the data either. Is there a way to coerce a DBA class object into a dataframe implemented in DiffBind? The person I'll be analyzing ChIPseq experiments for will need tables with differentially bound sites from his data comparisons so I need to learn how to get data out of DiffBind... 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] DESeq_1.10.1 lattice_0.20-10 locfit_1.5-8 [4] DiffBind_1.4.1 Biobase_2.18.0 GenomicRanges_1.10.5 [7] IRanges_1.16.4 BiocGenerics_0.4.0 BiocInstaller_1.8.3 loaded via a namespace (and not attached): [1] amap_0.8-7 annotate_1.36.0 AnnotationDbi_1.20.3 [4] DBI_0.2-5 edgeR_3.0.8 gdata_2.12.0 [7] genefilter_1.40.0 geneplotter_1.36.0 gplots_2.11.0 [10] grid_2.15.1 gtools_2.7.0 KernSmooth_2.23-8 [13] limma_3.14.4 RColorBrewer_1.0-5 RSQLite_0.11.2 [16] splines_2.15.1 stats4_2.15.1 survival_2.36-14 [19] tools_2.15.1 XML_3.95-0.1 xtable_1.7-0 [22] zlibbioc_1.4.0 El Mie, 30 de Enero de 2013, 13:28, Rory Stark escribi?: > > Hello- > > It looks like your sample sheet is fine. > > By default, if no contrasts are set up using dba.contrast, when you call > dba.analyze it attempts to add all the two-way contrasts between groups > where there are at least three samples in each group. In your case, > nothing meets these conditions, as there are only two "Resistant" samples. > > If you add a call to dba.contrast before the call to dba.analyze, you will > get the contrast you desire: > >> chippy = dba.contrast(chippy, minMembers=2) > > or, slightly more explicitly: > >> chippy = dba.contrast(chippy, categories=DBA_CONDITION, minMembers=2) > > or, even more explicitly: > >> chippy = dba.contrast(chippy, group1=chippy$masks$Resistant, group2 = >>chippy$masksResponsive, name1="Resistant", name2="Responsive") > > then: > >> chippy = dba.analyze(chippy) > > Cheers- > Rory > Hi, By default, dba.contrast won't create contrasts with less than 3 members in each group. The easiest solution is to set minMembers=2 when calling dba.contrast: > chippy = dba.contrast(chippy, categories=DBA_CONDITION, minMembers=2) Or you can create the contrast explicitly: > chippy = dba.contrast(chippy, group1=chippy$masks$Resistant, >group2=chippy$masks$Responsive, name1="a name", name2="another name") Hope this helps... Cheers, - Gord > > On Tue, 29 Jan 2013 15:21:23 +0100, jluis.lavin at unavarra.es wrote: > > >> >>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_peak >>s.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 >> >>We may monitor all incoming and outgoing emails in line with current >>legislation. We have taken steps to ensure that this email and >>attachments are free from any virus, but it remains your responsibility >>to ensure that viruses do not adversely affect you. >>Cancer Research UKRegistered charity in England and Wales (1089464), >>Scotland (SC041666) and the Isle of Man (1103) >>A company limited by guarantee. Registered company in England and Wales >>(4325234) and the Isle of Man (5713F). >>Registered Office Address: Angel Building, 407 St John Street, London >>EC1V 4AD. > > -- 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

@Rory, Thank you very much for your help, it works fine. I think I'll follow your advice about how to present the results to the researcher, it's a wise choice. ;-) El Mie, 30 de Enero de 2013, 16:09, Rory Stark escribi?: > Hi again- > > You need to explicitly retrieve the results of the analysis > (differentially bound sites) using dba.report: > >> chippy.db = dba.report(chippy) > > By default this will come back as a GRanges object. You can get it back as > a data frame using the DataType parameter: > >> chippy.db = dba.report(chippy, DataType = DBA_DATA_FRAME) > > > (note you can set the default data type by setting > chippy$config$DataType=DBA_DATA_FRAME). > > You can save the report to a csv file directly by specifying a file name > to dba.report: > >> chippy.db = dba.report(chippy,file="ChIPseq_diffbind") > > Which will return the report as a Granges object and create a file called > "ChIPseq_diffbind.csv". > > Note that you can control the contents of the report with other parameters > to dba.report. By default, it will include sites with an FDR < 0.10; for > each site you will get the interval, the log2 mean concentration of > normalized reads overall and in the two groups, the log2 fold change > (positive or negative depending on which group has a greater read > concentration), as well as the p-value and FDR (corrected for multiple > testing). You can change the threshold using the "th" and "fold" > parameters (setting th=1 will include all sites in the report, sorted by > lowest FDR). You can also include the counts for each sample (normalized > or non-normalized) by setting bCounts=TRUE, among other options. > > I usually return all the sites as a csv file, with counts for each sample, > to the biologist and let them think about cutoffs, using: > >> chippy.db = dba.report(chippy, th=1, bCounts=T, file"dba_chippy") > > > Cheers- > Rory > > > On 30/01/2013 14:51, "jluis.lavin at unavarra.es" <jluis.lavin at="" unavarra.es=""> > wrote: > >>Hello again, >> >>Thank you very much to Rory and Gordon for your kind and accurate help. >>Changing the minMembers parameter everything seemed to work fine and I've >>been able to perform the next steps of the whole analysis. >> >>Now I'm struggling with the retrieval of the analysis data; when I try to >>write the results into a .csv table with a simple command I get the >>following error: >> >>> write.csv( chippy, file="ChIPseq_diffbind.csv" ) >> >> >>Error in as.data.frame.default(x[[i]], optional = TRUE, stringsAsFactors >> = >>stringsAsFactors) : >> cannot coerce class '"DBA"' into a data.frame >> >>I read DiffBind's vignette and manual and I found the "save" command, but >>it doesn't allow me to retrieve the data either. >>Is there a way to coerce a DBA class object into a dataframe implemented >>in DiffBind? >>The person I'll be analyzing ChIPseq experiments for will need tables >> with >>differentially bound sites from his data comparisons so I need to learn >>how to get data out of DiffBind... >> >>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] DESeq_1.10.1 lattice_0.20-10 locfit_1.5-8 >>[4] DiffBind_1.4.1 Biobase_2.18.0 GenomicRanges_1.10.5 >>[7] IRanges_1.16.4 BiocGenerics_0.4.0 BiocInstaller_1.8.3 >> >>loaded via a namespace (and not attached): >> [1] amap_0.8-7 annotate_1.36.0 AnnotationDbi_1.20.3 >> [4] DBI_0.2-5 edgeR_3.0.8 gdata_2.12.0 >> [7] genefilter_1.40.0 geneplotter_1.36.0 gplots_2.11.0 >>[10] grid_2.15.1 gtools_2.7.0 KernSmooth_2.23-8 >>[13] limma_3.14.4 RColorBrewer_1.0-5 RSQLite_0.11.2 >>[16] splines_2.15.1 stats4_2.15.1 survival_2.36-14 >>[19] tools_2.15.1 XML_3.95-0.1 xtable_1.7-0 >>[22] zlibbioc_1.4.0 >> >> >>El Mie, 30 de Enero de 2013, 13:28, Rory Stark escribi?: >>> >>> Hello- >>> >>> It looks like your sample sheet is fine. >>> >>> By default, if no contrasts are set up using dba.contrast, when you >>>call >>> dba.analyze it attempts to add all the two-way contrasts between groups >>> where there are at least three samples in each group. In your case, >>> nothing meets these conditions, as there are only two "Resistant" >>>samples. >>> >>> If you add a call to dba.contrast before the call to dba.analyze, you >>>will >>> get the contrast you desire: >>> >>>> chippy = dba.contrast(chippy, minMembers=2) >>> >>> or, slightly more explicitly: >>> >>>> chippy = dba.contrast(chippy, categories=DBA_CONDITION, minMembers=2) >>> >>> or, even more explicitly: >>> >>>> chippy = dba.contrast(chippy, group1=chippy$masks$Resistant, group2 = >>>>chippy$masksResponsive, name1="Resistant", name2="Responsive") >>> >>> then: >>> >>>> chippy = dba.analyze(chippy) >>> >>> Cheers- >>> Rory >>> >>Hi, >> >>By default, dba.contrast won't create contrasts with less than 3 members >>in each group. The easiest solution is to set minMembers=2 when calling >>dba.contrast: >> >>> chippy = dba.contrast(chippy, categories=DBA_CONDITION, minMembers=2) >> >>Or you can create the contrast explicitly: >> >>> chippy = dba.contrast(chippy, group1=chippy$masks$Resistant, >>>group2=chippy$masks$Responsive, name1="a name", name2="another name") >> >>Hope this helps... >> >>Cheers, >> >> - Gord >>> >>> On Tue, 29 Jan 2013 15:21:23 +0100, jluis.lavin at unavarra.es wrote: >>> >>> >>>> >>>>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_pea >>>>ks >>>>.bed >>>>Contrl2 Neural K9 Resistant 2 Control2.bed Control_input.bed Control2_pe >>>>ak >>>>s.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 >>>> >>>>We may monitor all incoming and outgoing emails in line with current >>>>legislation. We have taken steps to ensure that this email and >>>>attachments are free from any virus, but it remains your responsibility >>>>to ensure that viruses do not adversely affect you. >>>>Cancer Research UKRegistered charity in England and Wales (1089464), >>>>Scotland (SC041666) and the Isle of Man (1103) >>>>A company limited by guarantee. Registered company in England and >>>> Wales >>>>(4325234) and the Isle of Man (5713F). >>>>Registered Office Address: Angel Building, 407 St John Street, London >>>>EC1V 4AD. >>> >>> >> >> >>-- >>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. 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