retrieve an averaged spectrum

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retrieve an averaged spectrum

July 03, 2012, 04:15:11 PM

Hello XCMS gurus,

I am running some infusions of standard compounds and am trying to use the getSpec() function to collect an averaged spectrum for a range of about 30 seconds during which time my standard is eluting.

test<-xcmsRaw("120703_601.CDF", profstep=0.01)
test
spec<-getSpec(test, rtrange=c(45:90))
spec2<-spec[order(spec[,2], decreasing=TRUE),]
spec2[1:20,]

mz intensity
[1,] 132.10220 84482.79
[2,] 132.10226 84476.03
[3,] 132.10229 84468.90
[4,] 132.10216 84434.43
[5,] 132.10213 84392.98
[6,] 132.10240 84370.49
[7,] 132.10243 84337.78
[8,] 132.10246 84293.82
[9,] 132.10205 84216.37
[10,] 132.10262 83990.52
[11,] 132.10196 83972.10
[12,] 132.10170 83246.16
[13,] 86.09711 37450.49
[14,] 86.09715 37449.09
[15,] 86.09710 37446.60
[16,] 86.09720 37443.11
[17,] 86.09707 37436.68
[18,] 86.09725 37416.95
[19,] 86.09694 37371.84
[20,] 86.09693 37367.13

As you can see, I am not getting any averaging of the spectra unless the mass matches exactly, though the documentation for getScan suggests it can be used for averaging multiple scans. Am I missing a parameter setting somewhere (a ppm error, for example), or am I trying to do something the function wasn't designed for? Any advice is greatly appreciated!

Corey

ps. as a secondary but relevent question: any way to subtract background spectrum from a portion of the infusion before the compound elutes?

Re: retrieve an averaged spectrum

Reply #1 – July 05, 2012, 02:18:07 PM

I have made a little progress using this code:

> xs <- xcmsSet(method="MSW", files="120703_library_test601.CDF", snthresh=1)
120703_library_test601:
> xsg<-group(xs, method="mzClust", mzppm=10, minfrac=5)
47.37 97.37 100.00
> peaklist <- peakTable(xsg, value="into")
> mz<-vector()
> intensity<-vector()
> for (i in 1:length(xsg@groupidx)) {
+ mz<-c(mz, mean(peaklist[xsg@groupidx[], "mz"]))
+ intensity<-c(intensity, mean(peaklist[xsg@groupidx[], "into"]))
+ }
> mz
[1] 86.09696 132.10216
> intensity
[1] 33563.66 67670.72
>

However, I seem to have lost my isotopes and lower abundance peaks. I have tried adjusting the snthresh values, to no avail. Pointers?
Corey

Re: retrieve an averaged spectrum

Reply #2 – July 27, 2012, 12:10:01 PM

I really haven't made any progress - any one have any tips? I just to average a few spectra from infusion data to increase spectrum quality. Thanks,
Corey

Re: retrieve an averaged spectrum

Reply #3 – July 28, 2012, 04:17:23 AM

Hi,

one thing that'd come to mind is to use group.mzClust()
which is an alignment of individual spectra. Originally developed
to group() data from direct infusion spectra in an xcmSet.

Then you could do intensity-weighted averages of the grouped peaks

Check out xcms/R/mzClust.R.

yours,
Steffen

Re: retrieve an averaged spectrum

Reply #4 – August 01, 2012, 11:42:42 AM

I think your approach is OK, the problem is that you have to optimize MSW peak picking first, which can be a little tricky, see also here.

Alternatively, you could export the spectra in centroid mode using your vendor software/converter or try the "deprofiling" code that was posted recently.

With the centroided/picked spectra you can use mzClust to align and postprocess the aligned data to get your averaged spectrum.
Some decision making will be necessary there, e.g. do you want to weight m/z by intensity, do you want the average for intensities or the local maxima, a noise filter based on the number of peaks aligned, etc.

Ralf

Re: retrieve an averaged spectrum

Reply #5 – February 19, 2013, 03:08:56 PM

Ralf,

I have spectra that are already centroided. I can extract individual spectra from the file using getSpec(), and I can choose those spectra by using the xcmsRaw()@TIC slot. I am trying to understand how to apply mzClust to to spectra. The default output for getScan is not an xcmsSet or Raw object structure, so I don't think I can use that directly as input for mzClust. If I could coerce a spectrum into an xcmsSet object structure, maybe this is possible.

So I think that I either need to
1. extract individal scans from a raw data file so that they are inserted into an xcmsSet object or
2. coerce the getSpec() output into something mzClust can take.

I am a bit stumped as to how to do this. Any tips? Thanks,

Corey

Re: retrieve an averaged spectrum

Reply #6 – February 20, 2013, 03:20:22 AM

Hi,

Quote from: "cbroeckl"

how to apply mzClust to to spectra.

Check out the mzClustGeneric in xcms/R/mzClust.R,
this is the function doing the actual work.

This is called from group(method="mzClust") and takes a simple mz/sample matrix.

Code: [Select]


          groups <- mzClustGeneric(peaks[,c("mz","sample")],
                                   sampclass=classlabel,
                                   mzppm=mzppm,mzabs=mzabs,
                                   minsamp=minsamp,
                                   minfrac=minfrac)

Quote from: "cbroeckl"

So I think that I either need to
1. extract individal scans from a raw data file so that they are inserted into an xcmsSet object or

This would be a new method for findPeaks(), which would be quite welcome!

For direct infusion with several scans (as opposed to single scans, which MSW expects)
I could envision that it is not too unlike centWave,
but without the wavelets (because there are no gaussian peak shapes):

It'd take a (very narrow) scanrange, detect ROIs, but skip the wavelet analysis,
and then apply one of the mzCenterFun to get a more accurate m/z than
you can expect from a single scan. A question would be whether and how
to integrate the intensities (Max ? Avg ?). Does that make sense ?

Yours,
Steffen

Re: retrieve an averaged spectrum

Reply #7 – February 20, 2013, 10:20:28 AM

Thanks for the tip Steffen

Stupid questions maybe: I have never really accessed the behind the scenes code, and for the life of me I cannot find the file you refer to. In my xcms/R folder there are only xcms documents (three of them), and my search isn't finding any files called mzClust.R on my C: drive. Sorry to be a pain.

Corey

Re: retrieve an averaged spectrum

Reply #8 – February 20, 2013, 04:43:08 PM

Hi,

Quote from: "cbroeckl"

Stupid questions
Corey

There are no stupid questions!

Quote from: "cbroeckl"

I have never really accessed the behind the scenes code, and for the life of me I cannot find the file you refer to.
Corey

For Windows (and Mac ?) the package downloads are "compiled", that's why you don't see them.
Linux will download the sources and compile on-the-fly during installation.

The source code is in the "package source" e.g. xcms_1.34.0.tar.gz at http://bioconductor.org/packages/releas ... /xcms.html
Alternatively, you cen check it out with SVN from https://hedgehog.fhcrc.org/bioconductor ... acks/xcms/
Check out http://bioconductor.org/developers/source-control/ for the password.

If you made any changes to the sources, you then will need to
R CMD INSTALL /path/to/xcmssources/
and re-load xcms into R.

Yours,
Steffen

Re: retrieve an averaged spectrum

Reply #9 – February 21, 2013, 02:37:58 PM

steffen,

THanks for the tips. I used the links you provided, and in the mzClust.R file under R, there is code:

Code: [Select]

mzClustGeneric <- function(p,sampclass=NULL,
                           mzppm = 20,
                           mzabs = 0,
                           minsamp = 1,
                           minfrac=0.5)

I did find the code snippet you quoted in the xcmsSet.R file.

Is it safe to assume that everything necessary to run xcmsSet() from the R console is located in the xcmsSet.R file? If not, how do I recognize calls to other files? Thanks again, being able to see all the details should allow me to understand everything (including how to properly write R code) better.
Corey