---
title: "LCMS data preprocessing and analysis with xcms"
package: xcms
output:
  BiocStyle::html_document:
    toc_float: true
vignette: >
  %\VignetteIndexEntry{LCMS data preprocessing and analysis with xcms}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
  %\VignetteDepends{xcms,RColorBrewer,faahKO,pander,magrittr}
  %\VignettePackage{xcms}
  %\VignetteKeywords{mass spectrometry, metabolomics}
bibliography: references.bib
csl: biomed-central.csl
references:
- id: dummy
  title: no title
  author:
  - family: noname
    given: noname
---

```{r biocstyle, echo = FALSE, results = "asis" }
BiocStyle::markdown()
```

**Package**: `r Biocpkg("xcms")`<br />
**Authors**: Johannes Rainer<br />
**Modified**: `r file.info("xcms.Rmd")$mtime`<br />
**Compiled**: `r date()`

```{r init, message = FALSE, echo = FALSE, results = "hide" }
## Silently loading all packages
library(BiocStyle)
library(xcms)
library(faahKO)
library(pander)
## Use socket based parallel processing on Windows systems
## if (.Platform$OS.type == "unix") {
##     register(bpstart(MulticoreParam(2)))
## } else {
##     register(bpstart(SnowParam(2)))
## }
register(SerialParam())
```


# Introduction

This documents describes data import, exploration, preprocessing and analysis of
LCMS experiments with `xcms` version >= 3. The examples and basic workflow was
adapted from the original *LC/MS Preprocessing and Analysis with xcms* vignette
from Colin A. Smith.

The new user interface and methods use the `XCMSnExp` object (instead of the
*old* `xcmsSet` object) as a container for the pre-processing results. To
support packages and pipelines relying on the `xcmsSet` object, it is however
possible to convert an `XCMSnExp` into a `xcmsSet` object using the `as` method
(i.e.`xset <- as(x, "xcmsSet")`, with `x` being an `XCMSnExp` object.


# Data import

`xcms` supports analysis of LC/MS data from files in (AIA/ANDI) NetCDF,
mzML/mzXML and mzData format. For the actual data import Bioconductor's
`r Biocpkg("mzR")` is used. For demonstration purpose we will analyze a
subset of the data from [@Saghatelian04] in which the metabolic consequences of
knocking out the fatty acid amide hydrolase (FAAH) gene in mice was
investigated. The raw data files (in NetCDF format) are provided with the
`faahKO` data package. The data set consists of samples from the spinal cords of
6 knock-out and 6 wild-type mice. Each file contains data in centroid mode
acquired in positive ion mode form 200-600 m/z and 2500-4500 seconds.

Below we load all required packages, locate the raw CDF files within the `faahKO`
package and build a *phenodata* data frame describing the experimental setup.

```{r load-libs-pheno, message = FALSE }
library(xcms)
library(faahKO)
library(RColorBrewer)
library(pander)
library(magrittr)

## Get the full path to the CDF files
cdfs <- dir(system.file("cdf", package = "faahKO"), full.names = TRUE,
            recursive = TRUE)
## Create a phenodata data.frame
pd <- data.frame(sample_name = sub(basename(cdfs), pattern = ".CDF",
                                   replacement = "", fixed = TRUE),
                 sample_group = c(rep("KO", 6), rep("WT", 6)),
                 stringsAsFactors = FALSE)
```

Subsequently we load the raw data as an `OnDiskMSnExp` object using the
`readMSData` method from the `MSnbase` package. While the `MSnbase` package was
originally developed for proteomics data processing, many of its functionality,
including raw data import and data representation, can be shared and reused in
metabolomics data analysis. Also, `MSnbase` can be used to *centroid*
profile-mode MS data (see the corresponding vignette in the `MSnbase` package).

```{r load-with-msnbase, message = FALSE }
raw_data <- readMSData(files = cdfs, pdata = new("NAnnotatedDataFrame", pd),
                       mode = "onDisk")
```

The `OnDiskMSnExp` object contains general information about the number of
spectra, retention times, the measured total ion current etc, but does not
contain the full raw data (i.e. the m/z and intensity values from each measured
spectrum). Its memory footprint is thus rather small making it an ideal object
to represent large metabolomics experiments while still allowing to perform
simple quality controls, data inspection and exploration as well as data
sub-setting operations. The m/z and intensity values are imported from the raw
data files on demand, hence the location of the raw data files should not be
changed after initial data import.


# Initial data inspection

The `OnDiskMSnExp` organizes the MS data by spectrum and provides the methods
`intensity`, `mz` and `rtime` to access the raw data from the files (the measured
intensity values, the corresponding m/z and retention time values). In addition,
the `spectra` method could be used to return all data encapsulated in `Spectrum`
classes. Below we extract the retention time values from the object.

```{r data-inspection-rtime, message = FALSE }
head(rtime(raw_data))
```

All data is returned as one-dimensional vectors (a numeric vector for `rtime`
and a `list` of numeric vectors for `mz` and `intensity`, each containing the
values from one spectrum), even if the experiment consists of multiple
files/samples. The `fromFile` function returns a numeric vector that provides
the mapping of the values to the originating file. Below we use the `fromFile`
indices to organize the `mz` values by file.

```{r data-inspection-mz, message = FALSE }
mzs <- mz(raw_data)

## Split the list by file
mzs_by_file <- split(mzs, f = fromFile(raw_data))

length(mzs_by_file)
```

As a first evaluation of the data we plot below the base peak chromatogram (BPC)
for each file in our experiment. We use the `chromatogram` method and set the
`aggregationFun` to `"max"` to return for each spectrum the maximal intensity
and hence create the BPC from the raw data. To create a total ion chromatogram
we could set `aggregationFun` to `sum`.

```{r data-inspection-bpc, message = FALSE, fig.align = "center", fig.width = 12, fig.height = 6 }
## Get the base peak chromatograms. This reads data from the files.
bpis <- chromatogram(raw_data, aggregationFun = "max")
## Define colors for the two groups
group_colors <- paste0(brewer.pal(3, "Set1")[1:2], "60")
names(group_colors) <- c("KO", "WT")

## Plot all chromatograms.
plot(bpis, col = group_colors[raw_data$sample_group])
```

The `chromatogram` method returned a `Chromatograms` object that organizes
individual `Chromatogram` objects (which in fact contain the chromatographic
data) in a two-dimensional array: columns represent samples and rows
(optionally) m/z and/or retention time ranges. Below we extract the chromatogram
of the first sample and access its retention time and intensity values.

```{r data-inspection-chromatogram, message = FALSE }
bpi_1 <- bpis[1, 1]
head(rtime(bpi_1))
head(intensity(bpi_1))
```

The `chromatogram` method supports also extraction of chromatographic data from
a m/z-rt slice of the MS data. In the next section we will use this method to
create an extracted ion chromatogram (EIC) for a selected peak.

Note that `chromatogram` reads the raw data from each file to calculate the
chromatogram. The `bpi` and `tic` methods on the other hand do not read any data
from the raw files but use the respective information that was provided in the
header definition of the input files (which might be different from the actual
data).

Below we create boxplots representing the distribution of total ion currents per
file. Such plots can be very useful to spot problematic or failing MS runs.

```{r  data-inspection-tic-boxplot, message = FALSE, fig.align = "center", fig.width = 8, fig.height = 4, fig.cap = "Distribution of total ion currents per file." }
## Get the total ion current by file
tc <- split(tic(raw_data), f = fromFile(raw_data))
boxplot(tc, col = group_colors[raw_data$sample_group],
        ylab = "intensity", main = "Total ion current")
```


# Chromatographic peak detection

Next we perform the chromatographic peak detection using the *centWave*
algorithm [@Tautenhahn:2008fx]. Before running the peak detection it is however
strongly suggested to visually inspect e.g. the extracted ion chromatogram of
internal standards or known compounds to evaluate and adapt the peak detection
settings since the default settings will not be appropriate for most LCMS
experiments. The two most critical parameters for *centWave* are the `peakwidth`
(expected range of chromatographic peak widths) and `ppm` (maximum expected
deviation of m/z values of centroids corresponding to one chromatographic peak;
this is usually much larger than the ppm specified by the manufacturer)
parameters.  To evaluate the typical chromatographic peak width we plot the EIC
for one peak.

```{r peak-detection-plot-eic, message = FALSE, fig.align = "center", fig.width = 8, fig.height = 5, fig.cap = "Extracted ion chromatogram for one peak." }
## Define the rt and m/z range of the peak area
rtr <- c(2700, 2900)
mzr <- c(334.9, 335.1)
## extract the chromatogram
chr_raw <- chromatogram(raw_data, mz = mzr, rt = rtr)
plot(chr_raw, col = group_colors[chr_raw$sample_group])
```

Note that `Chromatogram` objects extracted by the `chromatogram` method contain
an `NA` value if in a certain scan (i.e. for a specific retention time) no
signal was measured in the respective mz range. This is reflected by the lines
not being drawn as continuous lines in the plot above.

The peak above has a width of about 50 seconds. The `peakwidth` parameter should
be set to accommodate the expected widths of peak in the data set. We set it to
`20,80` for the present example data set.

For the `ppm` parameter we extract the full MS data (intensity, retention time
and m/z values) corresponding to the above peak. To this end we first filter the
raw object by retention time, then by m/z and finally plot the object with`type =
"XIC"` to produce the plot below. We use the *pipe* (`%>%`) command better
illustrate the corresponding workflow.

```{r peak-detection-plot-ms-data, message = FALSE, warning = FALSE, fig.aligh = "center", fig.width = 14, fig.height = 14, fig.cap = "Visualization of the raw MS data for one peak. For each plot: upper panel: chromatogram plotting the intensity values against the retention time, lower panel m/z against retention time plot. The individual data points are colored according to the intensity." }
raw_data %>%
    filterRt(rt = rtr) %>%
    filterMz(mz = mzr) %>%
    plot(type = "XIC")
```

In the present data there is actually no variation in the m/z values. Usually
one would see the m/z values (lower panel) scatter around the *real* m/z value
of the compound. The first step of the *centWave* algorithm defines so called
regions of interest (ROI) based on the difference of m/z values from consecutive
scans. In detail, m/z values from consecutive scans are included into a ROI if
the difference between the m/z and the mean m/z of the ROI is smaller than the
user defined `ppm` parameter. A reasonable choice for the `ppm` could thus be
the maximal m/z difference of data points from neighboring scans/spectra that
are part of the chromatographic peak. It is suggested to inspect the ranges of
m/z values for many compounds (either internal standards or compounds known to
be present in the sample) and define the `ppm` parameter for *centWave*
according to these.

Below we perform the chromatographic peak detection using the `findChromPeaks`
method. The submitted *parameter* object defines which algorithm will be used
and allows to define the settings for this algorithm. Note that we set the
argument `noise` to `5000` to reduce the run time of this vignette. With this
setting we consider only signals with a value larger than 5000 in the peak
detection step.

```{r peak-detection-centwave, message = FALSE, results = "hide" }
cwp <- CentWaveParam(peakwidth = c(20, 80), noise = 5000)
xdata <- findChromPeaks(raw_data, param = cwp)
```

The results are returned as an `XCMSnExp` object which extends the
`OnDiskMSnExp` object by storing also LC/GC-MS preprocessing results. This means
also that all methods to sub-set and filter the data or to access the (raw) data
are inherited from the `OnDiskMSnExp` object. The results from the
chromatographic peak detection can be accessed with the `chromPeaks` method.

```{r peak-detection-chromPeaks, message = FALSE }
head(chromPeaks(xdata))
```

The returned `matrix` provides the m/z and retention time range for each
identified chromatographic peak as well as the integrated signal intensity
("into") and the maximal peak intensitity ("maxo"). Columns "sample" contains
the index of the sample in the object/experiment in which the peak was
identified.

Below we use the data from this table to calculate some per-file summaries.

```{r peak-detection-peaks-per-sample, message = FALSE, results = "asis" }
summary_fun <- function(z) {
    c(peak_count = nrow(z), rt = quantile(z[, "rtmax"] - z[, "rtmin"]))
}
T <- lapply(split.data.frame(chromPeaks(xdata),
                             f = chromPeaks(xdata)[, "sample"]),
            FUN = summary_fun)
T <- do.call(rbind, T)
rownames(T) <- basename(fileNames(xdata))
pandoc.table(T,
             caption = paste0("Summary statistics on identified chromatographic",
                              " peaks. Shown are number of identified peaks per",
                              " sample and widths/duration of chromatographic ",
                              "peaks."))
```

We can also plot the location of the identified chromatographic peaks in the
m/z - retention time space for one file using the `plotChromPeaks`
function. Below we plot the chromatographic peaks for the 3rd sample.

```{r peak-detection-chrom-peaks-plot, message = FALSE, fig.align = "center", fig.width = 8, fig.height = 8, fig.cap = "Identified chromatographic peaks in the m/z by retention time space for one sample." }
plotChromPeaks(xdata, file = 3)
```

To get a global overview of the peak detection we can plot the frequency of
identified peaks per file along the retention time axis. This allows to identify
time periods along the MS run in which a higher number of peaks was identified
and evaluate whether this is consistent across files.

```{r peak-detection-chrom-peak-image, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 8, fig.cap = "Frequency of identified chromatographic peaks along the retention time axis. The frequency is color coded with higher frequency being represented by yellow-white. Each line shows the peak frequency for one file." }
plotChromPeakImage(xdata)
```

Next we highlight the identified chromatographic peaks for the example peak from
before. Evaluating such plots on a list of peaks corresponding to known peaks or
internal standards helps to ensure that peak detection settings were appropriate
and correctly identified the expected peaks.

```{r peak-detection-highlight-chrom-peaks-plot, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 8, fig.cap = "Signal for an example peak. Red and blue colors represent KO and wild type samples, respectively. The rectangles indicate the identified chromatographic peaks per sample." }
plot(chr_raw, col = group_colors[chr_raw$sample_group], lwd = 2)
highlightChromPeaks(xdata, border = group_colors[chr_raw$sample_group],
                    lty = 3, rt = rtr, mz = mzr, type = "rect")
```

In the plot above the peak definitions are indicated with rectangles containing
the full peak. In addition it is possible to represent the apex position of each
peak with a single point (passing argument`type = "point"` to the function), or
draw the actually identified peak by specifying `type = "polygon"`. Below we use
this option to fill the peak area for each identified chromatographic peak in
each sample. Whether individual peaks can be still identified in such a plot
depends however on the number of samples from which peaks are drawn.

```{r peak-detection-highlight-chrom-peaks-plot-polygon, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 8, fig.cap = "Signal for an example peak. Red and blue colors represent KO and wild type samples, respectively. The signal area of identified chromatographic peaks are filled with a color." }
plot(chr_raw, col = group_colors[chr_raw$sample_group], lwd = 2)
highlightChromPeaks(xdata, col = group_colors[chr_raw$sample_group],
                    lty = 3, rt = rtr, mz = mzr, border = NA,
                    type = "polygon")
```

Note that we can also specifically extract identified chromatographic peaks for
a selected region by providing the respective m/z and retention time ranges with
the `mz` and `rt` arguments in the `chromPeaks` method.

```{r peak-detection-chrom-peak-table-selected, message = FALSE, results = "asis" }
pander(chromPeaks(xdata, mz = mzr, rt = rtr),
       caption = paste("Identified chromatographic peaks in a selected ",
                       "m/z and retention time range."))
```

Finally we plot also the distribution of peak intensity per sample. This allows
to investigate whether systematic differences in peak signals between samples
are present.

```{r peak-detection-chrom-peak-intensity-boxplot, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 8, fig.cap = "Peak intensity distribution per sample." }
## Extract a list of per-sample peak intensities (in log2 scale)
ints <- split(log2(chromPeaks(xdata)[, "into"]),
              f = chromPeaks(xdata)[, "sample"])
boxplot(ints, varwidth = TRUE, col = group_colors[xdata$sample_group],
        ylab = expression(log[2]~intensity), main = "Peak intensities")
grid(nx = NA, ny = NULL)
```


# Alignment

The time at which analytes elute in the chromatography can vary between samples
(and even compounds). Such a difference was already observable in the extracted
ion chromatogram plot shown as an example in the previous section. The alignment
step, also referred to as retention time correction, aims at adjusting this by
shifting signals along the retention time axis to align the signals between
different samples within an experiment.

A plethora of alignment algorithms exist (see [@Smith:2013gr]), with some of
them being implemented also in `xcms`. The method to perform the
alignment/retention time correction in `xcms` is `adjustRtime` which uses
different alignment algorithms depending on the provided parameter class. In the
example below we use the *obiwarp* method [@Prince:2006jj] to align the
samples. We use a`binSize = 0.6` which creates warping functions in mz bins of 0.6. Also here
it is advisable to modify the settings for each experiment and evaluate if
retention time correction did align internal controls or known compounds
properly.

```{r alignment-obiwarp, message = FALSE, results = "hide" }
xdata <- adjustRtime(xdata, param = ObiwarpParam(binSize = 0.6))
```

`adjustRtime`, besides calculating adjusted retention times for each spectrum,
does also adjust the reported retention times of the identified chromatographic
peaks.

To extract the adjusted retention times we can use the `adjustedRtime` method,
or simply the `rtime` method that, if present, returns by default adjusted
retention times from an `XCMSnExp` object.

```{r alignment-rtime, message = FALSE }
## Extract adjusted retention times
head(adjustedRtime(xdata))

## Or simply use the rtime method
head(rtime(xdata))
```

*Raw* retention times can be extracted from an `XCMSnExp` containing
aligned data with `rtime(xdata, adjusted = FALSE)`.

To evaluate the impact of the alignment we plot the BPC on the adjusted data. In
addition we plot the differences of the adjusted- to the raw retention times per
sample using the `plotAdjustedRtime` function.

```{r alignment-obiwarp-plot, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 10, fig.cap = "Obiwarp aligned data. Base peak chromatogram after alignment (top) and difference between adjusted and raw retention times along the retention time axis (bottom)." }
## Get the base peak chromatograms.
bpis_adj <- chromatogram(xdata, aggregationFun = "max")
par(mfrow = c(2, 1), mar = c(4.5, 4.2, 1, 0.5))
plot(bpis_adj, col = group_colors[bpis_adj$sample_group])
## Plot also the difference of adjusted to raw retention time.
plotAdjustedRtime(xdata, col = group_colors[xdata$sample_group])
```

Too large differences between adjusted and raw retention times could indicate
poorly performing samples or alignment.

Alternatively we could use the *peak groups* alignment method that adjusts the
retention time by aligning previously identified *hook peaks* (chromatographic
peaks present in most/all samples). Ideally, these hook peaks should span most
part of the retention time range. Below we first restore the raw retention times
(also of the identified peaks) using the `dropAdjustedRtime` methods. Note that
a `drop*` method is available for each preprocessing step allowing to remove the
respective results from the `XCMSnExp` object.

```{r alignment-drop, message = FALSE }
## Does the object have adjusted retention times?
hasAdjustedRtime(xdata)

## Drop the alignment results.
xdata <- dropAdjustedRtime(xdata)

## Does the object have adjusted retention times?
hasAdjustedRtime(xdata)
```

**Note**: `XCMSnExp` objects hold the raw along with the adjusted retention
times and subsetting will in most cases drop the adjusted retention
times. Sometimes it might thus be useful to **replace** the raw retention times
with the adjusted retention times. This can be done with the
`applyAdjustedRtime`.

As noted above the *peak groups* method requires peak groups (features) present
in most samples to perform the alignment. We thus have to perform a first
correspondence run to identify such peaks (details about the algorithm used are
presented in the next section). We use here again default settings, but it is
strongly advised to adapt the parameters for each data set. The definition of
the sample groups (i.e. assignment of individual samples to the sample groups in
the experiment) is mandatory for the `PeakDensityParam`. If there are no sample
groups in the experiment `sampleGroups` should be set to a single value for each
file (e.g. `rep(1, length(fileNames(xdata))`).

```{r alignment-peak-groups, message = FALSE }
## Correspondence: group peaks across samples.
pdp <- PeakDensityParam(sampleGroups = xdata$sample_group,
                        minFraction = 0.8)
xdata <- groupChromPeaks(xdata, param = pdp)

## Now the retention time correction.
pgp <- PeakGroupsParam(minFraction = 0.85)

## Get the peak groups that would be used for alignment.
xdata <- adjustRtime(xdata, param = pgp)
```

Note also that we could use the `adjustedRtimePeakGroups` method on the object
before alignment to evaluate on which features (peak groups) the alignment would
be performed. This can be useful to test different settings for the peak groups
algorithm. Also, it is possible to manually select or define certain peak groups
(i.e. their retention times per sample) and provide this matrix to the
`PeakGroupsParam` class with the `peakGroupsMatrix` argument.

Below plot the difference between raw and adjusted retention times using the
`plotAdjustedRtime` function, which, if the *peak groups* method is used for
alignment, also highlights the peak groups used in the adjustment.

```{r alignment-peak-groups-plot, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 8, fig.cap = "Peak groups aligned data." }
## Plot the difference of adjusted to raw retention time.
plotAdjustedRtime(xdata, col = group_colors[xdata$sample_group],
                  peakGroupsCol = "grey", peakGroupsPch = 1)
```

At last we evaluate the impact of the alignment on the test peak.

```{r alignment-peak-groups-example-peak, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 10, fig.cap = "Example extracted ion chromatogram before (top) and after alignment (bottom)." }
par(mfrow = c(2, 1))
## Plot the raw data
plot(chr_raw, col = group_colors[chr_raw$sample_group])

## Extract the chromatogram from the adjusted object
chr_adj <- chromatogram(xdata, rt = rtr, mz = mzr)
plot(chr_adj, col = group_colors[chr_raw$sample_group])
```


# Correspondence

The final step in the metabolomics preprocessing is the correspondence that
matches detected chromatographic peaks between samples (and depending on the
settings, also within samples if they are adjacent). The method to perform the
correspondence in `xcms` is `groupChromPeaks`. We will use the *peak density*
method [@Smith:2006ic] to group chromatographic peaks. The algorithm combines
chromatographic peaks depending on the density of peaks along the retention time
axis within small slices along the mz dimension. To illustrate this we plot
below the chromatogram for an mz slice with multiple chromatographic peaks
within each sample. We use below a value of 0.4 for the `minFraction` parameter
hence only chromatographic peaks present in at least 40% of the samples per
sample group are grouped into a feature. The sample group assignment is
specified with the `sampleGroups` argument.

```{r correspondence-example-slice, message = FALSE, fig.align = "center", fig.width = 10, fig.height = 10, fig.cap = "Example for peak density correspondence. Upper panel: chromatogram for an mz slice with multiple chromatographic peaks. Middle and lower panel: identified chromatographic peaks at their retention time (x-axis) and index within samples of the experiments (y-axis) for different values of the bw parameter. The black line represents the peak density estimate. Grouping of peaks (based on the provided settings) is indicated by grey rectangles." }
## Define the mz slice.
mzr <- c(305.05, 305.15)

## Extract and plot the chromatograms
chr_mzr <- chromatogram(xdata, mz = mzr, rt = c(2500, 4000))
par(mfrow = c(3, 1), mar = c(1, 4, 1, 0.5))
cols <- group_colors[chr_mzr$sample_group]
plot(chr_mzr, col = cols, xaxt = "n", xlab = "")
## Highlight the detected peaks in that region.
highlightChromPeaks(xdata, mz = mzr, col = cols, type = "point", pch = 16)
## Define the parameters for the peak density method
pdp <- PeakDensityParam(sampleGroups = xdata$sample_group,
                        minFraction = 0.4, bw = 30)
par(mar = c(4, 4, 1, 0.5))
plotChromPeakDensity(xdata, mz = mzr, col = cols, param = pdp,
                     pch = 16, xlim = c(2500, 4000))
## Use a different bw
pdp <- PeakDensityParam(sampleGroups = xdata$sample_group,
                        minFraction = 0.4, bw = 20)
plotChromPeakDensity(xdata, mz = mzr, col = cols, param = pdp,
                     pch = 16, xlim = c(2500, 4000))
```

The upper panel in the plot above shows the extracted ion chromatogram for each
sample with the detected peaks highlighted. The middle and lower plot shows the
retention time for each detected peak within the different samples. The black
solid line represents the density distribution of detected peaks along the
retention times. Peaks combined into *features* (peak groups) are indicated with
grey rectangles. Different values for the `bw` parameter of the
`PeakDensityParam` were used:`bw = 30` in the middle and `bw = 20` in the lower
panel. With the default value for the parameter `bw` the two neighboring
chromatographic peaks would be grouped into the same feature, while with a `bw`
of 20 they would be grouped into separate features.  This grouping depends on
the parameters for the density function and other parameters passed to the
algorithm with the `PeakDensityParam`.

```{r correspondence, message = FALSE }
## Perform the correspondence
pdp <- PeakDensityParam(sampleGroups = xdata$sample_group,
                        minFraction = 0.4, bw = 20)
xdata <- groupChromPeaks(xdata, param = pdp)
```

The results from the correspondence can be extracted using the
`featureDefinitions` method, that returns a `DataFrame` with the definition of
the features (i.e. the mz and rt ranges and, in column `peakidx`, the index of
the chromatographic peaks in the `chromPeaks` matrix for each feature).

```{r correspondence-featureDefs, message = FALSE }
## Extract the feature definitions
featureDefinitions(xdata)
```

The `featureValues` method returns a `matrix` with rows being features and
columns samples. The content of this matrix can be defined using the `value`
argument. The default is`value = "index"` which simply returns the index of the
peak (in the `chromPeaks` matrix) assigned to a feature in a sample. Setting
`value = "into"` returns a matrix with the integrated signal of the peaks
corresponding to a feature in a sample. Any column name of the `chromPeaks`
matrix can be passed to the argument `value`. Below we extract the integrated
peak intensity per feature/sample.

```{r correspondence-feature-values, message = FALSE }
## Extract the into column for each feature.
head(featureValues(xdata, value = "into"))
```

This feature matrix contains `NA` for samples in which no chromatographic peak
was detected in the feature's m/z-rt region. While in many cases there might
indeed be no peak signal in the respective region, it might also be that there
is signal, but the peak detection algorithm failed to detect a chromatographic
peak. `xcms` provides the `fillChromPeaks` method to *fill in* intensity data
for such missing values from the original files. The *filled in* peaks are added
to the `chromPeaks` matrix and are flagged with an `1` in the `"is_filled"`
column. Below we perform such a filling-in of missing peaks.

```{r fill-chrom-peaks, message = FALSE }
## Filling missing peaks using default settings. Alternatively we could
## pass a FillChromPeaksParam object to the method.
xdata <- fillChromPeaks(xdata)

head(featureValues(xdata))
```

For features without detected peaks in a sample, the method extracts all
intensities in the mz-rt region of the feature, integrates the signal and adds a
*filled-in* peak to the `chromPeaks` matrix. No peak is added if no signal is
measured/available for the mz-rt region of the feature. For these, even after
filling in missing peak data, a `NA` is reported in the `featureValues` matrix.

It should be mentioned that `fillChromPeaks` uses columns `"rtmin"`, `"rtmax"`,
`"mzmin"` and `"mzmax"` from the `featureDefinitions` table to define the region
from which the signal should be integrated to generate the filled-in peak
signal. These values correspond however to the positions of the peak apex not
the peak boundaries of all chromatographic peaks assigned to a feature. It might
be advisable to increase this area in retention time dimension by a constant
value appropriate to the average peak width in the experiment. Such a value can
be specified with `fixedRt` of the `FillChromPeaksParam`. If the average peak
width in the experiment is 20 seconds, specifying`fixedRt = 10` ensures that the area
from which peaks are integrated is at least 20 seconds wide.

Below we compare the number of missing values before and after filling in
missing values. We can use the parameter `filled` of the `featureValues` method
to define whether or not filled-in peak values should be returned too.

```{r  fill-chrom-peaks-compare, message = FALSE }
## Missing values before filling in peaks
apply(featureValues(xdata, filled = FALSE), MARGIN = 2,
      FUN = function(z) sum(is.na(z)))

## Missing values after filling in peaks
apply(featureValues(xdata), MARGIN = 2,
      FUN = function(z) sum(is.na(z)))

```

Next we use the `featureSummary` function to get a general per-feature summary
that includes the number of samples in which a peak was found or the number of
samples in which more than one peak was assigned to the feature. Specifying also
sample groups breaks down these summary statistics for each individual sample
group.

```{r featureSummary, message = FALSE }
head(featureSummary(xdata, group = xdata$sample_group))
```

The performance of peak detection, alignment and correspondence should always be
evaluated by inspecting extracted ion chromatograms e.g. of known compounds,
internal standards or identified features in general. The `featureChromatograms`
function allows to extract chromatograms for each feature present in
`featureDefinitions`. The returned `Chromatograms` object contains an ion
chromatogram for each feature (each row containing the data for one feature) and
sample (each column representing containing data for one sample). Below we
extract the chromatograms for the first 4 features.

```{r featureChromatograms, message = FALSE }
feature_chroms <- featureChromatograms(xdata, features = 1:4)

feature_chroms
```

And plot the extracted ion chromatograms for feature number 3 and 4.

```{r feature-eic, message = FALSE, fig.align = "center", fig.width = 8, fig.height = 4, fig.cap = "Extracted ion chromatograms for feature 3 and 4." }
par(mfrow = c(1, 2))
plot(feature_chroms[3, ], col = group_colors[feature_chroms$sample_group])
plot(feature_chroms[4, ], col = group_colors[feature_chroms$sample_group])
```

At last we perform a principal component analysis to evaluate the grouping of
the samples in this experiment. Note that we did not perform any data
normalization hence the grouping might (and will) also be influenced by
technical biases.

```{r final-pca, message = FALSE, fig.align = "center", fig.width = 8, fig.height = 8, fig.cap = "PCA for the faahKO data set, un-normalized intensities." }
## Extract the features and log2 transform them
ft_ints <- log2(featureValues(xdata, value = "into"))

## Perform the PCA omitting all features with an NA in any of the
## samples. Also, the intensities are mean centered.
pc <- prcomp(t(na.omit(ft_ints)), center = TRUE)

## Plot the PCA
cols <- group_colors[xdata$sample_group]
pcSummary <- summary(pc)
plot(pc$x[, 1], pc$x[,2], pch = 21, main = "",
     xlab = paste0("PC1: ", format(pcSummary$importance[2, 1] * 100,
                                   digits = 3), " % variance"),
     ylab = paste0("PC2: ", format(pcSummary$importance[2, 2] * 100,
                                   digits = 3), " % variance"),
     col = "darkgrey", bg = cols, cex = 2)
grid()
text(pc$x[, 1], pc$x[,2], labels = xdata$sample_name, col = "darkgrey",
     pos = 3, cex = 2)

```

We can see the expected separation between the KO and WT samples on PC2. On PC1
samples separate based on their ID, samples with an ID <= 18 from samples with
an ID > 18. This separation might be caused by a technical bias
(e.g. measurements performed on different days/weeks) or due to biological
properties of the mice analyzed (sex, age, litter mates etc).


# Further data processing and analysis

Normalizing features' signal intensities is required, but at present not (yet)
supported in `xcms` (some methods might be added in near future). Also, for the
identification of e.g. features with significant different
intensities/abundances it is suggested to use functionality provided in other R
packages, such as Bioconductor's excellent `limma` package. To enable support
also for other packages that rely on the *old* `xcmsSet` result object, it is
possible to coerce the new `XCMSnExp` object to an `xcmsSet` object using
`xset <- as(x, "xcmsSet")`, with `x` being an `XCMSnExp` object.


# Additional details and notes

For a detailed description of the new data objects and changes/improvements
compared to the original user interface see the *new\_functionality* vignette.


## Evaluating the process history

`XCMSnExp` objects allow to capture all performed pre-processing steps along
with the used parameter class within the `@processHistory` slot. Storing also
the parameter class ensures the highest possible degree of analysis
documentation and in future might enable to *replay* analyses or parts of it.
The list of all performed preprocessings can be extracted using the
`processHistory` method.

```{r processhistory, message = FALSE }
processHistory(xdata)
```

It is also possible to extract specific processing steps by specifying its
type. Available *types* can be listed with the `processHistoryTypes`
function. Below we extract the parameter class for the alignment/retention time
adjustment step.

```{r processhistory-select, message = FALSE }
ph <- processHistory(xdata, type = "Retention time correction")

ph
```

And we can also extract the parameter class used in this preprocessing step.

```{r processhistory-param, message = FALSE }
## Access the parameter
processParam(ph[[1]])

```


## Subsetting and filtering

`XCMSnEx` objects can be subsetted/filtered using the `[` method, or one of the
many `filter*` methods. All these methods aim to ensure that the data in the
returned object is consistent. This means for example that if the object is
subsetted by selecting specific spectra (by using the `[` method) all identified
chromatographic peaks are removed. Correspondence results (i.e. identified
features) are removed if the object is subsetted to contain only data from
selected files (using the `filterFile` method). This is because the
correspondence results depend on the files on which the analysis was performed -
running a correspondence on a subset of the files would lead to different
results.

As an exception, it is possible to force keeping adjusted retention times in the
subsetted object setting the `keepAdjustedRtime` argument to `TRUE` in any of
the subsetting methods.

Below we subset our results object the data for the files 2 and 4.

```{r subset-filterFile, message = FALSE }
subs <- filterFile(xdata, file = c(2, 4))

## Do we have identified chromatographic peaks?
hasChromPeaks(subs)
```

Peak detection is performed separately on each file, thus the subsetted object
contains all identified chromatographic peaks from the two files. However, we
used a retention time adjustment (alignment) that was based on available
features. All features have however been removed and also the adjusted retention
times (since the alignment based on features that were identified on
chromatographic peaks on all files).

```{r subset-filterFile-2, message = FALSE }
## Do we still have features?
hasFeatures(subs)

## Do we still have adjusted retention times?
hasAdjustedRtime(subs)
```

We can however use the `keepAdjustedRtime` argument to force keeping the
adjusted retention times.

```{r subset-filterFile-3, message = FALSE }
subs <- filterFile(xdata, keepAdjustedRtime = TRUE)

hasAdjustedRtime(subs)
```

The `filterRt` method can be used to subset the object to spectra within a
certain retention time range.

```{r subset-filterRt, message = FALSE }
subs <- filterRt(xdata, rt = c(3000, 3500))

range(rtime(subs))
```

Filtering by retention time does not change/affect adjusted retention times
(also, if adjusted retention times are present, the filtering is performed
**on** the adjusted retention times).

```{r subset-filterRt-2, message = FALSE }
hasAdjustedRtime(subs)
```

Also, we have all identified chromatographic peaks within the specified
retention time range:

```{r subset-filterRt-3, message = FALSE }
hasChromPeaks(subs)

range(chromPeaks(subs)[, "rt"])
```

The most natural way to subset any object in R is with `[`. Using `[` on an
`XCMSnExp` object subsets it keeping only the selected spectra. The index `i`
used in `[` has thus to be an integer between 1 and the total number of spectra
(across all files). Below we subset `xdata` using both `[` and `filterFile` to
keep all spectra from one file.

```{r subset-bracket, message = FALSE, warning = FALSE }
## Extract all data from the 3rd file.
one_file <- filterFile(xdata, file = 3)

one_file_2 <- xdata[fromFile(xdata) == 3]

## Is the content the same?
all.equal(spectra(one_file), spectra(one_file_2))
```

While the spectra-content is the same in both objects, `one_file` contains also
the identified chromatographic peaks while `one_file_2` does not. Thus, in most
situations subsetting using one of the filter functions is preferred over the
use of `[`.

```{r subset-bracket-peaks, message = FALSE }
## Subsetting with filterFile preserves chromatographic peaks
head(chromPeaks(one_file))

## Subsetting with [ not
head(chromPeaks(one_file_2))
```

Note however that also `[` does support the `keepAdjustedRtime` argument. Below
we subset the object to spectra 20:30.

```{r subset-bracket-keepAdjustedRtime, message = FALSE, warnings = FALSE }
subs <- xdata[20:30, keepAdjustedRtime = TRUE]

hasAdjustedRtime(subs)

## Access adjusted retention times:
rtime(subs)

## Access raw retention times:
rtime(subs, adjusted = FALSE)
```

As with `MSnExp` and `OnDiskMSnExp` objects, `[[` can be used to extract a
single spectrum object from an `XCMSnExp` object. The retention time of the
spectrum corresponds to the adjusted retention time if present.

```{r subset-double-bracket, message = FALSE }
## Extract a single spectrum
xdata[[14]]
```

At last we can also use the `split` method that allows to split an `XCMSnExp`
based on a provided factor `f`. Below we split `xdata` per file. Using
`keepAdjustedRtime = TRUE` ensures that adjusted retention times are not
removed.

```{r subset-split, message = FALSE }
x_list <- split(xdata, f = fromFile(xdata), keepAdjustedRtime = TRUE)

lengths(x_list)

lapply(x_list, hasAdjustedRtime)
```

Note however that there is also a dedicated `splitByFile` method instead for
that operation, that internally uses `filterFile` and hence does e.g. not remove
identified chromatographic peaks. The method does not yet support the
`keepAdjustedRtime` parameter and thus removes by default adjusted retention
times.

```{r subset-split-by-file, message = FALSE }
xdata_by_file <- splitByFile(xdata, f = factor(1:length(fileNames(xdata))))

lapply(xdata_by_file, hasChromPeaks)
```


## Parallel processing

Most methods in `xcms` support parallel processing. Parallel processing is
handled and configured by the `BiocParallel` Bioconductor package and can be
globally defined for an R session.

Unix-based systems (Linux, macOS) support `multicore`-based parallel
processing. To configure it globally we `register` the parameter class. Note also
that `bpstart` is used below to initialize the parallel processes.

```{r multicore, message = FALSE, eval = FALSE }
register(bpstart(MulticoreParam(2)))
```

Windows supports only socket-based parallel processing:

```{r snow, message = FALSE, eval = FALSE }
register(bpstart(SnowParam(2)))
```

Note that `multicore`-based parallel processing might be buggy or failing on
 macOS. If so, the `DoparParam` could be used instead (requiring the `foreach`
package).

For other options and details see the vignettes from the `BiocParallel` package.


# References