Package: peakPantheR
Authors: Arnaud Wolfer

1 Introduction

The peakPantheR package is designed for the detection, integration and reporting of pre-defined features in MS files (e.g. compounds, fragments, adducts, …).

The Parallel Annotation is set to detect and integrate multiple compounds in multiple files in parallel and store results in a single object. It can be employed to integrate a large number of expected features across a dataset.

Using the faahKO raw MS dataset as an example, this vignette will:

  • Detail the Parallel Annotation concept
  • Apply the Parallel Annotation to a subset of pre-defined features in the faahKO dataset

1.1 Abbreviations

  • ROI: Regions Of Interest
    • reference RT / m/z windows in which to search for a feature
  • uROI: updated Regions Of Interest
    • modifed ROI adapted to the current dataset which override the reference ROI
  • FIR: Fallback Integration Regions
    • RT / m/z window to integrate if no peak is found
  • TIC: Total Ion Chromatogram
    • the intensities summed across all masses for each scan
  • EIC: Extracted Ion Chromatogram
    • the intensities summed over a mass range, for each scan

2 Parallel Annotation Concept

Parallel compound integration is set to process multiple compounds in multiple files in parallel, and store results in a single object.

To acheive this, peakPantheR will:

  1. load a list of expected RT / m/z ROI and a list of files to process
  2. initialise an output object with expected ROI and file paths
  3. first pass (without peak filling) on a subset of representative samples (e.g QC samples):
    • for each file, detect features in each ROI and keep highest intensity
    • determine peak statistics for each feature
    • store results + EIC for each ROI
  4. visual inspection of first pass results, update ROI:
    • diagnostic plots: all EICs, peak apex RT / m/z & peak width evolution
    • correct ROI (remove interfering feature, correct RT shift)
    • define fallback integration regions (FIR) if no feature is detected (median RT / m/z start and end of found features)
  5. initialise a new output object, with updated regions of interest (uROI) and fallback integration regions (FIR), with all samples
  6. second pass (with peak filling) on all samples:
    • for each file, detect features in each uROI and keep highest intensity
    • determine peak statistics for each feature
    • integrate FIR when no peaks are found
    • store results + EIC for each uROI
  7. summary statistics:
    • plot EICs, apex and peakwidth evolution
    • compare first and second pass
  8. return the resulting object and/or table (row: file, col: compound)

Diagram of the workflow and functions used for parallel annotation.

3 Parallel Annotation Example

We can target 2 pre-defined features in 6 raw MS spectra file from the faahKO package using peakPantheR_parallelAnnotation(). For more details on the installation and input data employed, please consult the Getting Started with peakPantheR vignette.

3.1 Input Data

First the paths to 3 MS file from the faahKO are located and used as input spectras. In this example these 3 samples are considered as representative of the whole run (e.g. Quality Control samples):

library(faahKO)
## file paths
input_spectraPaths  <- c(system.file('cdf/KO/ko15.CDF', package = "faahKO"),
                        system.file('cdf/KO/ko16.CDF', package = "faahKO"),
                        system.file('cdf/KO/ko18.CDF', package = "faahKO"))
input_spectraPaths
#> [1] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko15.CDF"
#> [2] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko16.CDF"
#> [3] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko18.CDF"

Two targeted features (e.g. compounds, fragments, adducts, …) are defined and stored in a table with as columns:

  • cpdID (numeric)
  • cpdName (character)
  • rtMin (sec)
  • rtMax (sec)
  • rt (sec, optional / NA)
  • mzMin (m/z)
  • mzMax (m/z)
  • mz (m/z, optional / NA)
# targetFeatTable
input_targetFeatTable <- data.frame(matrix(vector(), 2, 8, dimnames=list(c(), 
                        c("cpdID", "cpdName", "rtMin", "rt", "rtMax", "mzMin", 
                            "mz", "mzMax"))), stringsAsFactors=FALSE)
input_targetFeatTable[1,] <- c("ID-1", "Cpd 1", 3310., 3344.888, 3390., 
                                522.194778, 522.2, 522.205222)
input_targetFeatTable[2,] <- c("ID-2", "Cpd 2", 3280., 3385.577, 3440., 
                                496.195038, 496.2, 496.204962)
input_targetFeatTable[,c(3:8)] <- sapply(input_targetFeatTable[,c(3:8)], 
                                        as.numeric)
cpdID cpdName rtMin rt rtMax mzMin mz mzMax
ID-1 Cpd 1 3310 3344.888 3390 522.194778 522.2 522.205222
ID-2 Cpd 2 3280 3385.577 3440 496.195038 496.2 496.204962

Additional compound and spectra metadata can be provided but isn’t employed during the fitting procedure:

# spectra Metadata
input_spectraMetadata  <- data.frame(matrix(c("sample type 1", "sample type 2", 
                            "sample type 1"), 3, 1, 
                            dimnames=list(c(),c("sampleType"))),
                            stringsAsFactors=FALSE)
sampleType
sample type 1
sample type 2
sample type 1

3.2 Initialise and Run Parallel Annotation

A peakPantheRAnnotation object is first initialised with the path to the files to process (spectraPaths), features to integrate (targetFeatTable) and additional information and parameters such as spectraMetadata, uROI, FIR and if they should be used (useUROI=TRUE, useFIR=TRUE):

library(peakPantheR)
init_annotation <- peakPantheRAnnotation(spectraPaths = input_spectraPaths,
                        targetFeatTable = input_targetFeatTable,
                        spectraMetadata = input_spectraMetadata)

The resulting peakPantheRAnnotation object is not annotated, does not contain and use uROI and FIR

init_annotation
#> An object of class peakPantheRAnnotation
#>  2 compounds in 3 samples. 
#>   updated ROI do not exist (uROI)
#>   does not use updated ROI (uROI)
#>   does not use fallback integration regions (FIR)
#>   is not annotated

peakPantheR_parallelAnnotation() will run the annotation across files in parallel (if ncores >0) and return the successful annotations (result$annotation) and failures (result$failures):

# annotate files serially
annotation_result <- peakPantheR_parallelAnnotation(init_annotation, ncores=0, 
                                                    verbose=TRUE)
#> Processing 2 compounds in 3 samples:
#>   uROI:  FALSE
#>   FIR:   FALSE
#> ----- ko15 -----
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Check input, mzMLPath must be a .mzML
#> Reading data from 2 windows
#> Data read in: 2.05 secs
#> Warning: rtMin/rtMax outside of ROI; datapoints cannot be used for mzMin/mzMax calculation, approximate mz and returning ROI$mzMin and ROI$mzMax for ROI #1
#> Found 2/2 features in 0.03 secs
#> Peak statistics done in: 0 secs
#> Feature search done in: 2.99 secs
#> ----- ko16 -----
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Check input, mzMLPath must be a .mzML
#> Reading data from 2 windows
#> Data read in: 2.07 secs
#> Warning: rtMin/rtMax outside of ROI; datapoints cannot be used for mzMin/mzMax calculation, approximate mz and returning ROI$mzMin and ROI$mzMax for ROI #1
#> Warning: rtMin/rtMax outside of ROI; datapoints cannot be used for mzMin/mzMax calculation, approximate mz and returning ROI$mzMin and ROI$mzMax for ROI #2
#> Found 2/2 features in 0.03 secs
#> Peak statistics done in: 0 secs
#> Feature search done in: 2.81 secs
#> ----- ko18 -----
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Check input, mzMLPath must be a .mzML
#> Reading data from 2 windows
#> Data read in: 1.96 secs
#> Warning: rtMin/rtMax outside of ROI; datapoints cannot be used for mzMin/mzMax calculation, approximate mz and returning ROI$mzMin and ROI$mzMax for ROI #1
#> Warning: rtMin/rtMax outside of ROI; datapoints cannot be used for mzMin/mzMax calculation, approximate mz and returning ROI$mzMin and ROI$mzMax for ROI #2
#> Found 2/2 features in 0.03 secs
#> Peak statistics done in: 0 secs
#> Feature search done in: 2.76 secs
#> Annotation object cannot be reordered by sample acquisition date
#> ----------------
#> Parallel annotation done in: 10.25 secs
#>   0 failure(s)

# successful fit
nbSamples(annotation_result$annotation)
#> [1] 3
data_annotation   <- annotation_result$annotation
data_annotation
#> An object of class peakPantheRAnnotation
#>  2 compounds in 3 samples. 
#>   updated ROI do not exist (uROI)
#>   does not use updated ROI (uROI)
#>   does not use fallback integration regions (FIR)
#>   is annotated

# list failed fit
annotation_result$failures
#> [1] file  error
#> <0 rows> (or 0-length row.names)

3.3 Process Parallel Annotation Results

Based on the fit results, updated ROI (uROI) and fallback integration region (FIR) can be automatically determined using annotationParamsDiagnostic():

  • uROI are established as the min/max (rt and m/z) of the found peaks (+/- 5% in RT)
  • FIR are established as the median of found rtMin, rtMax, mzMin, mzMax
updated_annotation  <- annotationParamsDiagnostic(data_annotation, verbose=TRUE)
#> uROI will be set as mimimum/maximum of found peaks (+/-5% of ROI in retention time)
#> FIR will be calculated as the median of found "rtMin","rtMax","mzMin","mzMax"

# uROI now exist
updated_annotation
#> An object of class peakPantheRAnnotation
#>  2 compounds in 3 samples. 
#>   updated ROI exist (uROI)
#>   does not use updated ROI (uROI)
#>   does not use fallback integration regions (FIR)
#>   is annotated

outputAnnotationDiagnostic() will save to disk annotationParameters_summary.csv containing the original ROI and newly determined uROI and FIR for manual validation. Additionnaly a diagnostic plot for each compound is saved for reference and can be generated in parallel with the argument ncores:

# create a colourScale based on the sampleType
uniq_sType <- sort(unique(spectraMetadata(updated_annotation)$sampleType),
                    na.last=TRUE)
col_sType  <- unname( setNames(c('blue', 'red'),
                c(uniq_sType))[spectraMetadata(updated_annotation)$sampleType] )

# create a temporary location to save the diagnotic (otherwise provide the path
# to the selected location)
output_folder <- tempdir()

# output fit diagnostic to disk
outputAnnotationDiagnostic(updated_annotation, saveFolder=output_folder, 
                            savePlots=TRUE, sampleColour=col_sType, 
                            verbose=TRUE, ncores=2)

The data saved in annotationParameters_summary.csv is as follow:

Table continues below
cpdID cpdName X ROI_rt ROI_mz ROI_rtMin ROI_rtMax ROI_mzMin
ID-1 Cpd 1 | 3344.888 522.2 3310 3390 522.194778
ID-2 Cpd 2 | 3385.577 496.2 3280 3440 496.195038
Table continues below
ROI_mzMax X uROI_rtMin uROI_rtMax uROI_mzMin uROI_mzMax uROI_rt
522.205222 | 3305.75893 3411.43628 522.194778 522.205222 3344.888
496.204962 | 3337.37666 3462.44903 496.195038 496.204962 3385.577
uROI_mz X FIR_rtMin FIR_rtMax FIR_mzMin FIR_mzMax
522.2 | 3326.10635 3407.27265 522.194778 522.205222
496.2 | 3365.02386 3453.40496 496.195038 496.204962

Diagnostic plot for compound 1: The top panel is an overlay of the extracted EIC across all samples with the fitted curve as dotted line. The panel under the EIC represent each found peak RT peakwidth (rtMin, rtMax and apex marked as dot), ordered with the first sample at the top. The bottom 3 panels represent found RT (peakwidth), m/z (peakwidth) and peak area by run order, with the corresponding histograms to the right

ROI exported to .csv can be updated based on the diagnostic plots; uROI (updated ROI potentially used for all samples) and FIR (fallback integration regions for when no peak is found) can also be tweaked to better fit the peaks.

3.4 New Initialisation with Updated Parameters to be Applied to All Study Samples

Following this manual validation of the fit on reference samples, the modified parameters in the .csv file can be reloaded and applied to all study samples.

3.4.1 Load new fit parameters

peakPantheR_loadAnnotationParamsCSV() will load the new .csv parameters (as generated by outputAnnotationDiagnostic()) and initialise a peakPantheRAnnotation object without spectraPaths, spectraMetadata or cpdMetadata which will need to be added before annotation. useUROI and useFIR are set to FALSE by default and will need to be modified according to the analysis to run. uROIExist is established depending on the .csv uROI column, and will only be set to TRUE if no NA are present. It is possible to reset the FIR values with the uROI windows using resetFIR().

update_csv_path <- '/path_to_new_csv/'

# load csv
new_annotation <- peakPantheR_loadAnnotationParamsCSV(update_csv_path)
#> uROIExist set to TRUE
#> New peakPantheRAnnotation object initialised for 2 compounds

new_annotation
#> An object of class peakPantheRAnnotation
#>  2 compounds in 0 samples. 
#>   updated ROI exist (uROI)
#>   does not use updated ROI (uROI)
#>   does not use fallback integration regions (FIR)
#>   is not annotated

new_annotation <- resetFIR(new_annotation)
#> FIR will be reset with uROI values

3.4.2 Add new samples to process

Now that the fit parameters were set on 3 representative samples (e.g. QC), the same processing can be applied to all study samples. resetAnnotation() will reinitialise all the results and modify the samples or compounds targeted as required:

## new files
new_spectraPaths   <- c(system.file('cdf/KO/ko15.CDF', package = "faahKO"),
                        system.file('cdf/WT/wt15.CDF', package = "faahKO"),
                        system.file('cdf/KO/ko16.CDF', package = "faahKO"),
                        system.file('cdf/WT/wt16.CDF', package = "faahKO"),
                        system.file('cdf/KO/ko18.CDF', package = "faahKO"),
                        system.file('cdf/WT/wt18.CDF', package = "faahKO"))

new_spectraPaths
#> [1] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko15.CDF"
#> [2] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt15.CDF"
#> [3] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko16.CDF"
#> [4] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt16.CDF"
#> [5] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko18.CDF"
#> [6] "/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt18.CDF"

Below we define the metadata of these new samples:

## new spectra metadata
new_spectraMetadata  <- data.frame(matrix(c("KO", "WT", "KO", "WT", "KO", "WT"),
                                        6, 1, dimnames=list(c(), c("Group"))), 
                                    stringsAsFactors=FALSE)
Group
KO
WT
KO
WT
KO
WT 
## add new samples to the annotation loaded from csv, useUROI, useFIR

new_annotation <- resetAnnotation(new_annotation, spectraPaths=new_spectraPaths,
                                spectraMetadata=new_spectraMetadata, 
                                useUROI=TRUE, useFIR=TRUE)
#> peakPantheRAnnotation object being reset:
#>   Previous "ROI", "cpdID" and "cpdName" value kept
#>   Previous "uROI" value kept
#>   Previous "FIR" value kept
#>   Previous "cpdMetadata" value kept
#>   New "spectraPaths" value set
#>   New "spectraMetadata" value set
#>   Previous "uROIExist" value kept
#>   New "useUROI" value set
#>   New "useFIR" value set
new_annotation
#> An object of class peakPantheRAnnotation
#>  2 compounds in 6 samples. 
#>   updated ROI exist (uROI)
#>   uses updated ROI (uROI)
#>   uses fallback integration regions (FIR)
#>   is not annotated

3.5 Run Final Parallel Annotation

We can now run the final annotation on all samples with the optimised targeted features:

# annotate files serially
new_annotation_result <- peakPantheR_parallelAnnotation(new_annotation, 
                                                        ncores=0, verbose=FALSE)
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.
#> Polarity can not be extracted from netCDF files, please set manually the polarity with the 'polarity' method.

# successful fit
nbSamples(new_annotation_result$annotation)
#> [1] 6

final_annotation      <- new_annotation_result$annotation
final_annotation
#> An object of class peakPantheRAnnotation
#>  2 compounds in 6 samples. 
#>   updated ROI exist (uROI)
#>   uses updated ROI (uROI)
#>   uses fallback integration regions (FIR)
#>   is annotated

# list failed fit
new_annotation_result$failures
#> [1] file  error
#> <0 rows> (or 0-length row.names)

3.5.1 Output final results

The final fits can be saved to disk with outputAnnotationDiagnostic():

# create a colourScale based on the sampleType
uniq_group <- sort(unique(spectraMetadata(final_annotation)$Group),na.last=TRUE)
col_group  <- unname( setNames(c('blue', 'red'),
                    c(uniq_sType))[spectraMetadata(final_annotation)$Group] )

# create a temporary location to save the diagnotic (otherwise provide the path
# to the selected location)
final_output_folder <- tempdir()

# output fit diagnostic to disk
outputAnnotationDiagnostic(final_annotation, saveFolder=final_output_folder,
                        savePlots=TRUE, sampleColour=col_group, verbose=TRUE)

For each processed sample, a peakTables contains all the fit information for all compounds targeted. annotationTable( , column) will group the values across all samples and compounds for any peakTables column:

# peakTables for the first sample
peakTables(final_annotation)[[1]]
Table continues below
found rtMin rt rtMax mzMin mz mzMax peakArea
TRUE 3342 3342 3395 522.2 522.2 522.2 18409123
TRUE 3345 3387 3428 496.2 496.2 496.2 35467323
Table continues below
maxIntMeasured maxIntPredicted is_filled ppm_error rt_dev_sec
889280 907347 FALSE 0.02338 -2.928
1128960 1113682 FALSE 0.0246 0.9518
tailingFactor asymmetryFactor cpdID cpdName
203.5 377.4 ID-1 Cpd 1
1.005 1.009 ID-2 Cpd 2 
# Extract the found peak area for all compounds and all samples
annotationTable(final_annotation, column='peakArea')
Table continues below
  ID-1
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko15.CDF 18409123
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt15.CDF 23871264
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko16.CDF 24775525
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt16.CDF 25012332
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko18.CDF 21909568
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt18.CDF 21729136
  ID-2
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko15.CDF 35467323
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt15.CDF 37965512
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko16.CDF 37795145
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt16.CDF 34499235
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/KO/ko18.CDF 36717689
/home/biocbuild/bbs-3.11-bioc/R/library/faahKO/cdf/WT/wt18.CDF 36961319

Finally all annotation results can be saved to disk as .csv with outputAnnotationResult(). These .csv will contain the compound metadata, spectra metadata and a file for each column of peakTables (with samples as rows and compounds as columns):

# create a temporary location to save the diagnotic (otherwise provide the path
# to the selected location)
final_output_folder <- tempdir()

# save
outputAnnotationResult(final_annotation, saveFolder=final_output_folder, 
                        annotationName='ProjectName', verbose=TRUE)
#> Compound metadata saved at /final_output_folder/ProjectName_cpdMetadata.csv
#> Spectra metadata saved at 
#>     /final_output_folder/ProjectName_spectraMetadata.csv
#> Peak measurement "found" saved at /final_output_folder/ProjectName_found.csv
#> Peak measurement "rtMin" saved at /final_output_folder/ProjectName_rtMin.csv
#> Peak measurement "rt" saved at /final_output_folder/ProjectName_rt.csv
#> Peak measurement "rtMax" saved at /final_output_folder/ProjectName_rtMax.csv
#> Peak measurement "mzMin" saved at /final_output_folder/ProjectName_mzMin.csv
#> Peak measurement "mz" saved at /final_output_folder/ProjectName_mz.csv
#> Peak measurement "mzMax" saved at /final_output_folder/ProjectName_mzMax.csv
#> Peak measurement "peakArea" saved at 
#>     /final_output_folder/ProjectName_peakArea.csv
#> Peak measurement "maxIntMeasured" saved at 
#>     /final_output_folder/ProjectName_maxIntMeasured.csv
#> Peak measurement "maxIntPredicted" saved at 
#>     /final_output_folder/ProjectName_maxIntPredicted.csv
#> Peak measurement "is_filled" saved at 
#>     /final_output_folder/ProjectName_is_filled.csv
#> Peak measurement "ppm_error" saved at 
#>     /final_output_folder/ProjectName_ppm_error.csv
#> Peak measurement "rt_dev_sec" saved at 
#>     /final_output_folder/ProjectName_rt_dev_sec.csv
#> Peak measurement "tailingFactor" saved at 
#>     /final_output_folder/ProjectName_tailingFactor.csv
#> Peak measurement "asymmetryFactor" saved at 
#>     /final_output_folder/ProjectName_asymmetryFactor.csv
#> Summary saved at /final_output_folder/ProjectName_summary.csv