You want to remove the manual gates before call |gating| on the 
|auto_gating| object. Say the first manual gate was |singlet|, simply 
|Rm("singlet", auto_gating)| will do.

By the way, given that you might need to save the gating results later 
on anyway, instead of |clone|, you can use |archiving| functions to 
achieve the same:

|#save the manual gating set to a different disc copy
#Note that we use symbolic link option here for cdf file to avoid the unnecessary copy of flow data
#since they can be shared by both manual and auto gating sets
archive_dir <-"/your/directory/auto_gating/"
save_gs(gating_set, path = archive_dir, cdf ="link")

#load it back to a different gatingset object
auto_gs <- load_gs(archive_dir)
Rm("singlet", auto_gs)

#do the auto gating
...

#re-save it
save_gs(auto_gs, archive_dir, overwrite =TRUE)
|

Mike

On 06/21/2014 06:27 PM, Eisa Mahyari wrote:

> Hi Mike,
>
> Thank you for your answer and it worked! However, now that it finished 
> automated gating of my data I want to plot it, thus I used as an example:
>
> plotGate(auto_gating, c("CD4"), path = "auto", projections = 
> list("CD4" = c(x = "CD8", y = "CD4")), gpar = c(nrow = 2))
>
>
> But the gate shown in the figure is the manual gate; even though I use 
> the variable auto_gating not gating_set; the latter  is the variable 
> that has the manual gating.
>
> Does this has to with the fact that I used the clone function? Is 
> there another way to populate this variable or a manual gate is always 
> needed? Because if the manual gate has gates with  identical names, it 
> skips the gating as it says its already found a gate with that name.
>
> auto_gating<-clone(gating_set)
>
>
> Basically I want to show how it gated.
>
>
>
> Thank you,
> -Eisa
>
>
>
>
>
>
> From: Mike <wjiang2@fhcrc.org <mailto:wjiang2@fhcrc.org>>
> Date: Thu, 19 Jun 2014 13:46:31 -0700
> To: <rgottard@fhcrc.org <mailto:rgottard@fhcrc.org>>, Eisa Mahyari 
> <mahyarie@ohsu.edu <mailto:mahyarie@ohsu.edu>>, Greg Finak 
> <gfinak@fhcrc.org <mailto:gfinak@fhcrc.org>>, 
> <bioconductor@r-project.org <mailto:bioconductor@r-project.org>>
> Subject: Re: OpenCyto
>
> Eisa,
>
> As the error shows, you want to make sure the dimension used in 'pop' 
> and 'dim' column to be consistent, that said, you want to change 
> 'dims' from 'B4-A,B3-A' to ''CD4,CD8'.
>
> In general, I'd recommend you to use the partial marker name (e.g. 
> 'CD3') for `dims` column since it is more meaningful than channel 
> name. As long as this partial string is uniquely identifiable within 
> your flow data, openCyto should be able to match it without problem.
>
> Another suggestion is to use the partial ('Tcells') instead of full 
> path ('/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells'), again, 
> as long as it is uniquely identifiable within the gating tree, it 
> should further simplify your template.
>
> Let me know if it helps,
>
> Mike
>>
>>     On Wed, Jun 18, 2014 at 2:27 PM, Eisa Mahyari <mahyarie@ohsu.edu
>>     <mailto:mahyarie@ohsu.edu>> wrote:
>>
>>         Hi Dr. Gottardo,
>>
>>         My name is Eisa Mahyari and I am a graduate student down at
>>         OHSU. I am using OpenCyto and have hit some problems using it
>>         with the data I have available. I was wondering if I could
>>         troubleshoot it with someone in your lab; I would greatly
>>         appreciate the help. Below please note that I have attached
>>         my procedure.
>>
>>         Thank you in advance,
>>         Sincerely,
>>         Eisa
>>
>>
>>
>>         ##########
>>         #I load the xml made by flowjo
>>
>>         ws <-openWorkspace("HTflowR_easygate3.xml"); ws
>>         gating_set<-parseWorkspace(ws,name="DEMOTRY",path="sampledataset/DEMOFCS/",isNcdf=TRUE)
>>
>>         #load & attach annotations
>>
>>         #load the template (please see below; it is pasted as a csv)
>>         gt<-gatingTemplate("sampledataset/KCHS10451_140114/gt_080.man4.csv")
>>         auto_gating<-clone(gating_set)
>>         gating(x = gt, y = auto_gating)
>>
>>         The error I get is :
>>
>>         Error in FUN(gh, ...) :
>>           X,Y axis do not match between 'dims'(B4-A,B3-A) and
>>         'pop'(CD4+CD8-)
>>
>>         I have tried the other method of defining CD4 and 8 (I.e., *
>>         as the alias) as a first try, and that was not working.
>>
>>
>>         I have a simple FCS file with the colnames as (the person who
>>         collected the data did not name them correctly).
>>         "HDR-T" "FSC-A" "SSC-A" "V1-A"  "B1-A"  "B2-A"  "*_B3-A_*"
>>          "*B4-A*"  "R1-A"  "R2-A"
>>         But this list corresponds to:
>>         "time" "FSC-A" "SSC-A" "DumpPacBlueA" "IfngFITCA"
>>         "TNFaPEA" "*_CD8PercpCy55A_*" "*CD4PECy7A*" "IL2APCA"
>>         "CD3APCCy7A"
>>
>>         The template I made:
>>
>>         alias,pop,parent,dims,gating_method,gating_args,collapseDataForGating,groupBy,preprocessing_method,preprocessing_args
>>         Boundry,Boundry,root,"FSC-A,SSC-A",boundary,"max=c(2.5e5,2.5e5)",,,,
>>         nonDebris,nonDebris+,/Boundry,FSC-A,mindensity,"gate_range=c(5e4,1e5),adjust=1.5",,,,
>>         DumpClean,DumpClean,/Boundry/nonDebris,V1-A,mindensity,"gate_range=c(10e2,10e4),adjust=1.5",,,,
>>         SmallLymphocytes,SmallLymphocytes,/Boundry/nonDebris/DumpClean,"FSC-A,SSC-A",flowClust,"K=2,quantile=0.95,target=c(1e5,5e4)",,,prior_flowClust,K=2
>>         Tcells,CD3+,/Boundry/nonDebris/DumpClean/SmallLymphocytes,R2-A,mindensity,"gate_range=c(10e3,10e4)",,,,
>>         CD8+,CD8+,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells,B3-A,mindensity,"gate_range=c(200,9000)",,,,
>>         CD8-,CD8-,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells,B3-A,mindensity,"gate_range=c(-5000,190)",,,,
>>         CD4+,CD4+,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells,B4-A,mindensity,"gate_range=c(200,9000)",,,,
>>         CD4-,CD4-,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells,B4-A,mindensity,"gate_range=c(-5000,190)",,,,
>>         CD4,CD4+CD8-,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells,"B4-A,B3-A",refGate,CD4+:CD8-,,,,
>>         CD8,CD4-CD8+,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells,"B4-A,B3-A",refGate,CD4-:CD8+,,,,
>>         TNFa,TNFa,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells/CD8,B2-A,cytokine,"adjust=2,tol=1e-4",TRUE,PTID:VISITNO,,
>>         TNFa,TNFa,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells/CD4,B2-A,cytokine,"adjust=2,tol=1e-4",TRUE,PTID:VISITNO,,
>>         IL2,IL2,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells/CD8,R1-A,cytokine,"adjust=2,tol=1e-4",TRUE,PTID:VISITNO,,
>>         IL2,IL2,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells/CD4,R1-A,cytokine,"adjust=2,tol=1e-4",TRUE,PTID:VISITNO,,
>>         IFNg,IFNg,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells/CD8,B1-A,cytokine,"adjust=2,tol=1e-4",TRUE,PTID:VISITNO,,
>>         IFNg,IFNg,/Boundry/nonDebris/DumpClean/SmallLymphocytes/Tcells/CD4,B1-A,cytokine,"adjust=2,tol=1e-4",TRUE,PTID:VISITNO,,
>>
>>
>>
>>         R version 3.1.0 (2014-04-10)
>>         Platform: x86_64-apple-darwin10.8.0 (64-bit)
>>
>>         locale:
>>         [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
>>
>>         attached base packages:
>>         [1] grid      stats graphics  grDevices utils datasets
>>          methods   base
>>
>>         other attached packages:
>>         [1] data.table_1.9.2  openCyto_1.2.4  flowWorkspace_3.10.08
>>         gridExtra_0.9.1
>>         [5] ncdfFlow_2.10.30  flowViz_1.28.21 lattice_0.20-29
>>         flowCore_1.30.7
>>
>>         loaded via a namespace (and not attached):
>>          [1] Biobase_2.24.0  BiocGenerics_0.10.0 car_2.0-20  clue_0.3-48
>>          [5] cluster_1.15.2  coda_0.16-1 corpcor_1.6.6 DEoptimR_1.0-1
>>          [9] fda_2.4.3 flowClust_3.4.10  flowStats_3.22.4  graph_1.42.0
>>         [13] gtools_3.4.1  hexbin_1.26.3 IDPmisc_1.1.17
>>          KernSmooth_2.23-12
>>         [17] ks_1.9.2  latticeExtra_0.6-26 MASS_7.3-33 Matrix_1.1-3
>>         [21] MCMCpack_1.3-3  misc3d_0.8-4  mvoutlier_2.0.4
>>         mvtnorm_0.9-99992
>>         [25] nnet_7.3-8  parallel_3.1.0  pcaPP_1.9-49          pls_2.4-3
>>         [29] plyr_1.8.1  R.methodsS3_1.6.1 R.oo_1.18.0 R.utils_1.32.4
>>         [33] RBGL_1.40.0 RColorBrewer_1.0-5  Rcpp_0.11.2 reshape2_1.4
>>         [37] rgl_0.93.996  Rgraphviz_2.8.1 robCompositions_1.8.0
>>         robustbase_0.91-1
>>         [41] rrcov_1.3-4 sgeostat_1.0-25 stats4_3.1.0  stringr_0.6.2
>>         [45] tools_3.1.0 XML_3.98-1.1  zlibbioc_1.10.0
>>
>>
>>
>>
>>
>>     -- 
>>     Raphael Gottardo, PhD
>>     Associate Member
>>     Vaccine and Infectious Disease Division
>>     Fred Hutchinson Cancer Research Center
>>     Phone: 206-667-4076 <tel:206-667-4076>
>>     Web: www.rglab.org <http://www.rglab.org>
>>
>>
>>
>>
>> -- 
>> Raphael Gottardo, PhD
>> Associate Member
>> Vaccine and Infectious Disease Division
>> Fred Hutchinson Cancer Research Center
>> Phone: 206-667-4076
>> Web: www.rglab.org <http://www.rglab.org>
>

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