Dear List

I am beginner of R/bioconductor. I am analyzing a microarray dataset obtained from Affymetrix Porcine Gene 1.0 ST array. With the help of posts of the site, I have summarized the expression values for each array probes using  ‘oligo’ package in R v3.1.2.  However, I got every time the warning message below while RMA normalization. 

> eset <- oligo::rma(oligoRaw)

Background correcting

Normalizing

Calculating Expression

Warning message:

'isIdCurrent' is deprecated.

Use 'dbIsValid' instead.

See help("Deprecated") 

What does it mean? how can fix it?

While loading platform design package I got the warning message below-

> librarypd.porgene.1.0.st)

Warning message:

package ‘pd.porgene.1.0.st’ was built under R version 3.2.0 

Does this making wrong in downstream process? As i am using R v 3.1.2

Secondly, the big battle I faced in annotation and biological interpretation of the data. I tried with ‘porcine.db’ package for annotation but not sure is it the right one or not?

With limma package I can run the lmFit function and get topTable but that contains only Affy probeset  ID of eight digit number (e.g  ‘15192707’, ‘15212448’,…).  

How can I get the list of deferentially expressed genes with biological info like gene name, symbol, ensembl_id, fold change and adj p value ?  How can I filter genes which up and down regulated in contrast group of interest?

Please help me with the suggestions/tips/codes for way to go.  Thanks very much in advance.  

Here is the code i am trying with-

#load libraby

library(oligo)

library(limma)

library(porcine.db)

library(pd.porgene.1.0.st)


#nomalization

oligoRaw <- read.celfiles(list.celfiles())

eset <- oligo::rma(oligoRaw)

write.exprs(eset,file="data.normal.txt")


#linear model fit

design <- model.matrix(~ 0+factor(c(1,1,1,2,2,2,3,3,3, 4,4,4,5,5,5,6,6,6,7,7,7)))

colnames(design) <- c("cont.0h", "cont.6h", "vac.6h", "cont.24h", "vac.24h", "cont.72h", "vac.72h")

rownames(design)<-colnames(eset)

fit <- lmFit(eset, design)


# contrast groups for Diff expressed genes

cont.dif1<-makeContrasts(Dif24hr=(vac.24h-cont.0h), levels=design)

fit1 <- contrasts.fit(fit, cont.dif1)

fit.eBayes <- eBayes(fit1)

topTable(fit.eBayes, coef=1, adjust="BH")

results <- decideTests(fit.eBayes)

vennDiagram(results)


#Annotation

my_frame <- data.frame(exprs(eset))

porcine()

Annot <- data.frame(ACCNUM=sapply(contents(porcineACCNUM), paste, collapse=", "), SYMBOL=sapply(contents(porcineSYMBOL), paste, collapse=", "), DESC=sapply(contents(porcineGENENAME), paste, collapse=", "))

all <- merge(Annot, my_frame, by.x=0, by.y=0, all=T)

write.table(all,file="data.annoted.txt",sep="\t")

summary(all)

sessionInfo()

R version 3.1.2 (2014-10-31)
Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252   
[3] LC_MONETARY=English_United States.1252 LC_NUMERIC=C                          
[5] LC_TIME=English_United States.1252    

attached base packages:
[1] stats4    parallel  stats     graphics  grDevices utils     datasets  methods  
[9] base     

other attached packages:
 [1] genefilter_1.48.1        pd.porgene.1.0.st_3.10.0 porcine.db_3.0.0        
 [4] org.Ss.eg.db_3.0.0       RSQLite_1.0.0            DBI_0.3.1               
 [7] AnnotationDbi_1.28.1     GenomeInfoDb_1.2.3       limma_3.22.1            
[10] oligo_1.30.0             Biostrings_2.34.1        XVector_0.6.0           
[13] IRanges_2.0.1            S4Vectors_0.4.0          Biobase_2.26.0          
[16] oligoClasses_1.28.0      BiocGenerics_0.12.1     

loaded via a namespace (and not attached):
 [1] affxparser_1.38.0     affyio_1.34.0         annotate_1.44.0      
 [4] BiocInstaller_1.16.1  bit_1.1-12            codetools_0.2-9      
 [7] ff_2.2-13             foreach_1.4.2         GenomicRanges_1.18.3 
[10] iterators_1.0.7       preprocessCore_1.28.0 splines_3.1.2        
[13] survival_2.37-7       tools_3.1.2           XML_3.98-1.1         
[16] xtable_1.7-4          zlibbioc_1.12.0 

Please help me with the suggestions/tips/codes for way to go.  Thanks very much in advance.  

Sincerely

Amin

You don't have to worry about the warning for 'isIdCurrent' this has to do with some underlying code changes that won't affect your results.

It looks like you have a devel version of pd.porgene.1.0.st. You can fix that by doing

library(BiocInstaller)
biocLite("pd.porgene.1.0.st")

##Also do this

biocLite("pig.db0")

The annotation question is a bit more tricky. Ideally you would have a porgene10sttranscriptcluster.db package to annotate with, but we haven't built an annotation package for this array. That said, it isn't that difficult to build your own. You first need to go to the Affy website (requires a free registration) http://www.affymetrix.com/Auth/analysis/downloads/na34/wtgene/PorGene-1_0-st-v1.na34.sscr2.transcript.csv.zip

Unzip this file somewhere convenient.

Then go to this gist:

EDIT:  Now that's cool ^^^^^^^^^^^^^ Didn't expect that to happen.

probably the easiest thing to do is click the 'view raw' button at the bottom right of the code, then copy-paste into a text file, and save as makeTranscriptPkg.R, in the same directory that contains your PorGene-1_0-st-v1.na34.sscr2.transcript.csv file.

Now open R, change the directory to wherever you put those files, and source() the makeTranscriptPkg.R file. You can now build an annotation package by doing

pv <- parseCsvFiles("PorGene-1_0-st-v1.na34.sscr2.transcript.csv")

makePkg(pv$outname, pv$outsub, "8.2.1", "PIGCHIP_DB")

install.packages("porgene10sttranscriptcluster.db", repos = NULL, type = "source")

Hi James,

Great! Many thanks for the support. I could built my package with the code you provided and it worked well except a typo, should 'parseCsv' be 'parseCsvFiles' in the first command after running the source () function.

Best regards

Amin

Hi James and others,

I am curious to look for what comes out about alternate splicing events from my PorGene 1.0 ST array data set. After normalized with 'probeset' as target with oligo package, i could find some deferentially expressed probes/exons with limma, now i need to annotate them.

Could you please help me providing with the code for building package for exon level annotation (e.g. something like 'porgene10stprobeset.db').

Many thanks in advance.

Best regards

Amin

Hello,

I need to build an annotation package for  my GeneChip® Mouse Transcriptome Assay 1.0 array.

I followed  James' code but  it did not work for this platform. 

Would you please help  to make it work?

Thank you in advance.  

Anita

>pv <- parseCsvFiles("MTA-1_0.na34.1.mm10.transcript.csv")

>ls()

[1] "makePkg"       "parseCsvFiles" "pv"           

>pv
$outname
[1] "mta10"

$outsub
[1] "transcript"

>makePkg(pv$outname, pv$outsub, "r1", "MOUSECHIP_DB")

Error in sqliteSendQuery(con, statement, bind.data) : error in statement: table metadata already exists

James, 
Another error message.
Would you please help?
Thank you.
Anita 

>unlink(dir(".", "db$|sqlite$"), recursive = TRUE)

> makePkg(pv$outname, pv$outsub, "r1", "MOUSECHIP_DB")
baseMapType is gb or gbNRef

Error in sqliteSendQuery(con, statement, bind.data) : error in statement: no such table: src.accession

In addition: Warning message: 'dbBeginTransaction' is deprecated. Use 'dbBegin' instead. See help("Deprecated")