Help with RAPIDR package

[PandoraMid]

Hi all! I need your help with an R package named RAPIDR. I'm new to use this package, but I really need it, but I don't know how I can perform my analysis without errors. Now I explain my steps.

1) I create a binned file with the following code:

> makeBinnedCountsFile(bam.file.list=c(bam1.bam, bam2.bam,bam3.bam,bam4.bam), sampleIDs=c("001", "003", "005", "013"), binned.counts.fname="output.csv", k=20000)

2) I create a reference with the following code:

> rapidr.dir<-system.file(package="RAPIDR")
> data(outcomes)
> data(gcContent)
> T21.pos<-which(outcomes$Dx=="T21")
> chr.lens<-sapply(gcContent, length)
> chr.names<-names(chr.lens)
> header<-c("SampleID")
> for(i in 1:length(chr.lens)){
+ header<-c(header,rep(chr.names[i], chr.lens[i]))}
> nbins<-sum(chr.lens)
> ncols<-nbins+1
> binned.counts<-matrix(nrow=nrow(outcomes), ncol=ncols)
> for(i in 1:nrow(binned.counts)){
+ binned.counts[i,]<-rpois(ncols, lambda=100)
+ if(i%in%T21.pos){
+ binned.counts[i,139087:141493]<-rpois(chr.lens[21], lambda=115)
+ }}
> binned.counts[,1]<-outcomes$SampleID
> colnames(binned.counts)<-header
> t<-tempfile()
> write.table(binned.counts, file=t, col.names=TRUE, row.names=FALSE, quote=FALSE, sep=",")
> "output.csv"<-t
> message(t)
/tmp/RtmpwrkRt5/file33292c2c486
> gcContent.fname<-paste(rapidr.dir, "/media/martina/VERBATIM HD/Altamedica/RAPIDR_0.1.1/RAPIDR/data/gcContent.rda", sep="")
> head(outcomes)
  SampleID     Dx Gender
1     1000 Normal Female
2     1001 Normal Female
3     1002    T21 Female
4     1003 Normal   Male
5     1004 Normal   Male
6     1005 Normal   Male
> ref.set<-createReferenceSetFromCounts("/media/martina/VERBATIM HD/risultati/output.csv", outcomes, gcCorrect=FALSE, PCA=FALSE, filterBin=FALSE, gcContentFile=gcContent.fname)

But I receive this error:

Loading binned counts file
Checking every sampleID has an outcome
No outcomes for Sample 001
No outcomes for Sample 003
No outcomes for Sample 005
No outcomes for Sample 013
Error in `[.data.frame`(sampleIDs.with.outcomes, , "Gender") : 
  undefined columns selected

The BAM files is from 2 male and 2 female, without aneuplodies and they come from ION Torrent, but I used the fastq file and re-alignd with bowtie2, and coverted into sorted-bam file with samtools.


Why I receive this error? How can I perform the analysis? Is there anyone who have some experience with this package and can help me?

Thank you

[Manonathan]

Sample Id in outcomes data frame should correspond to the sample file name . The reason for the error may be the change in sample Id .check whether both are having same name. If not, modifythe sample id or rename the sample file and do the process again.

[PandoraMid]

Hi! Thanks for your reply! I have a doubt: the only ID for sample that I create is in the creation of binned file. Where I can do this check? Is the second part of my code correct? Can I see all 3 aneuploidies with this code?

I'm sorry for my question, but I'm new with this kind of analysis!

[Manonathan]

Hi,
No issue. Me too struggled a lot to understand the workflow.

create a new text file with three columns like that in the outcomes data frame
Use sampleid, Gender and Dx.Use your sample files id, gender and Dx details and save it as "outcome.txt".Use the Female/Male term as it is case sensitive.
convert that into a data frame using
outcome<-read.table("outcome.txt",header=T)

head(outcome)

should be similar to the outcomes dataframe.

use this newly created dataframe in your script instead of outcomes.

[PandoraMid]

So, if I understood I have to create manually the outcomes, and then use this file with the function

createReferenceSetFromCounts

with: my binned file, my outcome file (made by output.txt and your instuction), gcCorrect and the others things?

Second question: is it ok if I create my outcomes file with csv extension?

Third question: do you use RAPIDR-Plus too?

[PandoraMid]

Hi! I tryed to use your tips but I can't resolve the situation. This is my outcome.txt file:

sampleID Gender Dx
001 Female Normal
003 Female Normal
005 Male Normal
013 Male Normal

Here there is the code that I use

> makeBinnedCountsFile(bam.file.list=c(bam1, bam2, bam3, bam4), sampleIDs=c("1", "3", "5", "13"), binned.counts.fname="/media/martina/VERBATIM HD/Altamedica/risultati/output_uffa.csv", k=20000)
Binning counts in bam files
doing the binning
Binning done in 42.556
doing the binning
Binning done in 31.375
doing the binning
Binning done in 49.443
doing the binning
Binning done in 35.978
> outcome<-read.table("/media/martina/VERBATIM HD/Altamedica/risultati/outcomes.txt", header=T)
> head(outcome)
  sampleID Gender     Dx
1        1 Female Normal
2        3 Female Normal
3        5   Male Normal
4       13   Male Normal
> ref.set<-createReferenceSetFromCounts(output.binned, outcome)
Loading binned counts file
Checking every sampleID has an outcome
No outcomes for Sample 1
No outcomes for Sample 3
No outcomes for Sample 5
No outcomes for Sample 13
Error in `[.data.frame`(sampleIDs.with.outcomes, , "Gender") : 
  undefined columns selected

I tryed to use "sampleid" "SampleID", "sampleid" but with the same results. Make I some errors?

[pooja.solanki2018]

I am getting the same error as above. Please anyone can help me regarding this issue

Error in `[.data.frame`(sampleIDs.with.outcomes, , "Gender") :
undefined columns selected