Warning problem after fillPeaks

Topic: Warning problem after fillPeaks (Read 8185 times) previous topic - next topic

Warning problem after fillPeaks

April 18, 2012, 02:38:54 AM

Hi all,
I have some problems after the fillPeaks. This is the code I work with:

xset <- xcmsSet(files, method="centWave", snthresh=10, ppm=10, mzdiff=0.01,
                prefilter=c(3,500), peakwidth=c(10,60), nSlaves=4)
idx<-which(xset@peaks[,"mz"] > 100 & xset@peaks[,"mz"] < 1000)
xset@peaks<-xset@peaks[idx,]
xsetR <- retcor(xset, method="obiwarp", profStep=0.1, plottype="deviation")
xsetR <- group(xsetR, bw=5, mzwid=0.025,  minsamp=5)
xsetF <- fillPeaks(xsetR)

I have this warning message after the fillPeaks step:

There were 50 or more warnings (use warnings() to see the first 50)
> warnings()
Warning messages:
1: In .local(object, ...) :
getPeaks: Peak m/z:397.366577148438-397.368316650391, RT:364.471-378.604is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

2: In .local(object, ...) :
getPeaks: Peak m/z:479.481033325195-479.483581542969, RT:364.471-391.31is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

3: In .local(object, ...) :
getPeaks: Peak m/z:480.484039306641-480.487976074219, RT:364.471-392.215is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

4: In .local(object, ...) :
getPeaks: Peak m/z:610.453125-610.457580566406, RT:364.471-383.212is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

5: In .local(object, ...) :
getPeaks: Peak m/z:617.063537597656-617.069519042969, RT:364.471-376.196is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

6: In .local(object, ...) :
getPeaks: Peak m/z:713.638122558594-713.643371582031, RT:364.471-398.001is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

7: In .local(object, ...) :
getPeaks: Peak m/z:798.143920898438-798.152557373047, RT:364.471-376.5195is out of retention time range for this sample (/home/cism/Documents/Controllo10.mzXML), using zero intensity value.

......

So, I think there is a specific problem, for example, in the file called Controllo10.mzXML, but clustering the RT differences it seems that the groups aren't too different.

Code: [Select]

minlength <- min(sapply(xsetR@rt$raw, length ))
devs <-
    sapply(1:length(xsetR@rt$corrected),
           function(x){
               (xsetR@rt$raw[[x]]-xsetR@rt$corrected[[x]])[1:minlength]})
colnames(devs) <- sampnames(xsetR)
ddevs <- dist(t(devs))
hdevs <- hclust(ddevs)
x11(); plot(hdevs)

The result of the clustering analysis is:

[attachment=1:29id13si]HCluster.png[/attachment:29id13si]

And this is the result of the retcor step:

[attachment=0:29id13si]RTDev.png[/attachment:29id13si]

Do you have any suggestion to try to solve this kind of problem?? Should I discard from my dataset the sample in the warning message??

Best
Riccardo

P.S. This enormous problem in the RT are due to the use of a HILIC LC column... :evil:

[attachment deleted by admin]

Re: Warning problem after fillPeaks

Reply #1 – April 18, 2012, 04:28:46 AM

Does this file otherwise look normal when you look at the chromatogram? This error suggests that there is no data above 6 mins for this file.

Re: Warning problem after fillPeaks

Reply #2 – April 18, 2012, 04:56:33 AM

Before I start work I check all the file using a Paul Benton's function:

Code: [Select]

CheckCDFfile <- function(file, type=".mzdata"){
    cat("n")
    cat(paste("Loading File:", file, sep=""))
    xr <- xcmsRaw(file, profstep=0)
    for(i in 1:length(xr@scanindex)){
        scan <- getScan(xr, scan=i)
        if(is.unsorted(scan[,"mz"]) == TRUE){
            cat(" x ")
            newfile <- sub(type, "-Fixed.mzdata",
                           file, ignore.case=TRUE, fixed=TRUE)
            write.mzdata(xr, newfile)
            file.copy(file, sub(type, ".OLD", file, ignore.case=TRUE))
            unlink(file)
            return(1)}
        if(i == length(xr@scanindex)){
            cat(" O ")
            return(0)}}}
###
sapply(files, CheckCDFfile)

All seems to be ok.

Re: Warning problem after fillPeaks

Reply #3 – April 18, 2012, 05:05:00 AM

This doesn't seem to check if the file got cut somehow if the file is still valid. I am just asking you to open the file in whatever viewer and check that the chromatogram has data in the whole retention time range as expected.

Re: Warning problem after fillPeaks

Reply #4 – April 18, 2012, 07:48:51 AM

I sow that all chromatograms "start" at 6 minutes (360s) and the problem is relative to a time window of RT:364.471-392.215. Could it be this the problem?

Moreover the files are ok, I visually checked them.

Re: Warning problem after fillPeaks

Reply #5 – April 18, 2012, 11:07:34 AM

Probably they start at slightly different times. So it cannot look for peaks identified very early in some samples if that retention time region is missing in others. I would just ignore...

You can try this (will probably take a while):

Code: [Select]

first_scan=c()
for (i in 1:length(files)){
first_scan[i] = min(xcmsRaw(files[i])@scantime)
}

hist(first_scan)

To see where your files actually start.

Re: Warning problem after fillPeaks

Reply #6 – April 19, 2012, 01:33:03 AM

Hi, using the code you suggested I obtain this:

[attachment=0:2zbklhg4]FirstScan.png[/attachment:2zbklhg4]

All the first scan is within the range in which fillPeaks works (i.e. ... Peak m/z:397.366577148438-397.368316650391, RT:364.471-378.604is out of retention time range for this sample...). So should I conclude that mass aren't present in that sample??

Best
Riccardo

[attachment deleted by admin]

Re: Warning problem after fillPeaks

Reply #7 – April 19, 2012, 02:38:15 AM

A peak was found in some sample in the range 364.471 - 378.604. As your graph shows not all files have data at early as 364.4, therefore the fillpeaks cannot attempt to find that peak in those files and is forced to set 0. I would just ignore it.

Re: Warning problem after fillPeaks

Reply #8 – April 23, 2012, 08:27:34 AM

Just to chime in: theres a good explanation which I copied over from the old wiki that steffen made. Link here:

viewtopic.php?f=25&t=148