[BioC] Bioconductor Digest, Vol 79, Issue 20

[Gordon K Smyth]

Dear Marcelo,

No, there isn't any bug.  limma simply expects the Signal Mean to be 
included as a column in the ImaGene output, which is the default behaviour 
of ImaGene.  You have suppressed the Signal Mean column, so when you use 
limma you have to specify the columns you want explicitly, which is what 
you have now done.  You took non-default action when you used ImaGene, so 
you need to take non-default action in limma.

Best wishes
Gordon

> Date: Sun, 20 Sep 2009 02:13:30 -0300
> From: Marcelo Laia <marcelolaia at gmail.com>
> Subject: [BioC] [limma] Possible bug on read.maimages function ?
> To: Bioconductor <bioconductor at stat.math.ethz.ch>
> Message-ID:
> 	<a35fc1810909192213x7b18b4b1yccb78cc11949d655 at mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> I think there is a bug on read.maimages limma function.
>
> I try:
>
>> targets <- readTargets()
>> files <- targets[,c("FileNameCy3","FileNameCy5")]
>> RG <- read.maimages(files, source="imagene")
> Read header information
> Read lamina1Cy3_551.txt
> Erro em `[.data.frame`(obj, , columns$f) : undefined columns selected
>
> After a lot of time, I try:
>
>> targets <- readTargets()
>> files <- targets[,c("FileNameCy3","FileNameCy5")]
>> RG <- read.maimages(files, source="imagene",columns=list(f="Signal Median",b="Background Median"))
> Read header information
> Read lamina1Cy3_551.txt
> Read lamina1Cy5_650.txt
> Read lamina2Cy3_551.txt
> Read lamina2Cy5_650.txt
> Read lamina3Cy3_532.txt
> Read lamina3Cy5_650.txt
> Read lamina5Cy3_532.txt
> Read lamina5Cy5_635.txt
>>
>
> Here are more information
>
>> targets
>  SlideNumber        FileNameCy3        FileNameCy5  Cy3       Cy5
> 1           1 lamina1Cy3_551.txt lamina1Cy5_650.txt Pra Banha
> 2           2 lamina2Cy3_551.txt lamina2Cy5_650.txt Pra Banha
> 3           3 lamina3Cy3_532.txt lamina3Cy5_650.txt Pra Banha
> 4           5 lamina5Cy3_532.txt lamina5Cy5_635.txt Pra Banha
>> files
>         FileNameCy3        FileNameCy5
> 1 lamina1Cy3_551.txt lamina1Cy5_650.txt
> 2 lamina2Cy3_551.txt lamina2Cy5_650.txt
> 3 lamina3Cy3_532.txt lamina3Cy5_650.txt
> 4 lamina5Cy3_532.txt lamina5Cy5_635.txt
>>
>
> Header of my files
>
> Begin Header
>
> 	version	8.0.1
>
> 	Date	Thu Jun 11 12:35:08 BRT 2009
>
> 	Image File	C:\Users\[snip]\lamina1Cy3_551.tif
>
> 	Page	0
>
> 	Page Name
>
> 	Inverted	false
>
> 	Begin Field Dimensions
>
> 		Field	Metarows	Metacols	Rows	Cols
>
> 		A	2	2	32	24
>
> 		B	2	2	32	24
>
> 	End Field Dimensions
>
> 	Begin Measurement parameters
>
> 		Segmentation Method	auto
>
> 		Signal Low	0.0
>
> 		Signal High	0.0
>
> 		Background Low	0.0
>
> 		Background High	0.0
>
> 		Background Buffer	2.0
>
> 		Background Width	5.0
>
> 	End Measurement parameters
>
> 	Begin Alerts
>
> 		Control Type	Minimum threshold	If tested	Percentage allowed	If
> failed	Maximum threshold	If tested	Percentage allowed	If failed	CV
> threshold	If tested	If failed
>
> 		BLANK	0.0	false	1.0%	false	500.0	true	0.1%	false	1.0	false	false
>
> 		POSITIVE	1000.0	true	0.1%	false	100000.0	false	1.0%	false	1.0	false	false
>
> 	End Alerts
>
> 	Begin Quality Flags
>
> 		Begin Flagging Settings
>
> 			Empty Spots	true	Threshold:	0.96
>
> 			Poor Spots	true
>
> 			Begin Poor Spots Parameters
>
> 				Background contamination flag	true	Threshold:	0.9995
>
> 				Background tested against subgrid data only	true
>
> 				Signal contamination flag	true	Threshold:	0.9995
>
> 				Signal contamination test connected to background contamination
> threshold	true
>
> 				Ignored percentage flag	true	Threshold:	25.2
>
> 				Open perimeter flag	true	Threshold:	20.0
>
> 				Shape regularity flag	true	Threshold:	0.6
>
> 				Area To Perimeter Ratio flag	false	Threshold:	0.7
>
> 				Offset flag	true	Threshold:	60.0
>
> 				Saturation flag	true
>
> 			End Poor Spots Parameters
>
> 			Negative Spots	true
>
> 		End Flagging Settings
>
> 		Begin Flagged spots
>
> 			# of Empty Spots: 2667
>
> 			# of Poor Spots: 288
>
> 			# of Negative Spots: 0
>
> 			# of Manually Flagged Spots: 0
>
> 		End Flagged spots
>
>
>
> 	End Quality Flags
>
> End Header
>
> Begin Raw Data
>
> 	Field	Meta Row	Meta Column	Row	Column	Gene ID	Annotation 1	Annotation
> 2	Flag	Signal Median	Background Median	Signal Mode	Background
> Mode	Signal Area	Background Area	Signal Total	Background Total	Signal
> Stdev	Background Stdev	Shape Regularity	Ignored Area	Spot Area	Ignored
> Median	Area To Perimeter	Open
> Perimeter	XCoord	YCoord	Diameter	Position offset	Offset X	Offset
> Y	Expected X	Expected Y	CM-X	CM-Y	CM Offset	CM Offset-X	CM
> Offset-Y	Min Diam	Max Diam	Control	Failed Control	Background
> contamination present	Signal contamination present	Ignored %
> failed	Open perimeter failed	Shape regularity failed	Perim-to-area
> failed	Offset failed	Empty spot	Negative spot	Selected spot	Saturated
> spot
>
>
>> sessionInfo()
> R version 2.9.2 (2009-08-24)
> i486-pc-linux-gnu
>
> locale:
> LC_CTYPE=pt_BR.UTF-8;LC_NUMERIC=C;LC_TIME=pt_BR.UTF-8;LC_COLLATE=pt_BR.UTF-8;LC_MONETARY=C;LC_MESSAGES=pt_BR.UTF-8;LC_PAPER=pt_BR.UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=pt_BR.UTF-8;LC_IDENTIFICATION=C
>
> attached base packages:
> [1] stats     graphics  grDevices utils     datasets  methods   base
>
> other attached packages:
> [1] limma_2.18.3
>
> loaded via a namespace (and not attached):
> [1] tools_2.9.2
>>
>
> Am I doing any mistake?
>
> Thank you very much
>
> -- 
> Marcelo Luiz de Laia
> Universidade do Estado de Santa Catarina
> UDESC - www.cav.udesc.br
> Lages - SC - Brazil
> Linux user number 487797