---
title: "Compounding (grouping) of LC-MS features"
package: xcms
output:
  BiocStyle::html_document:
    toc_float: true
vignette: >
  %\VignetteIndexEntry{LC-MS feature grouping}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
  %\VignetteDepends{xcms,msdata,BiocStyle,faahKO,pheatmap,MsFeatures}
  %\VignettePackage{xcms}
  %\VignetteKeywords{mass spectrometry, metabolomics}
---

```{r biocstyle, echo = FALSE, results = "asis"}
BiocStyle::markdown()
knitr::opts_chunk$set(echo = TRUE, warning = FALSE, message = FALSE)
```

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

```{r init, results = "hide", echo = FALSE}
## Silently loading all packages
library(BiocStyle)
library(xcms)
library(MsFeatures)
register(SerialParam())

```

# Introduction

In a typical LC-MS-based metabolomics experiment compounds eluting from the
chromatography are first ionized before being measured by mass spectrometry
(MS). During the ionization different (multiple) ions can be generated from the
same compound which all will be measured by MS. In general, the resulting data
is then pre-processed to identify chromatographic peaks in the data and to group
these across samples in the correspondence analysis. The result are distinct
LC-MS features, characterized by their specific m/z and retention time
range. Different ions generated during ionization will be detected as different
features. *Compounding* aims now at grouping such features presumably
representing signal from the same originating compound to reduce data set
complexity (and to aid in subsequent annotation steps). General MS feature
grouping functionality if defined by the `r Biocpkg("MsFeatures")` package with
additional functionality being implemented in the `xcms` package to enable the
compounding of LC-MS data.

This document provides a simple compounding workflow using `xcms`. Note that the
present functionality does not (yet) *aggregate* or combine the actual features
per values, but does only define the feature groups (one per compound).


# Compounding of LC-MS data

We demonstrate the compounding (feature grouping) functionality on the simple
toy data set used also in the `r Biocpkg("xcms")` package and provided through
the `faahKO` package. This data set consists of samples from 4 mice with
knock-out of the fatty acid amide hydrolase (FAAH) and 4 wild type
mice. Pre-processing of this data set is described in detail in the *xcms*
vignette of the `xcms` package. Below we load all required packages and the
result from this pre-processing updating also the location of the respective raw
data files on the current machine.

```{r load-data}
library(xcms)
library(faahKO)
library(MsFeatures)

data("xdata")
## Update the path to the files for the local system
dirname(xdata) <- c(rep(system.file("cdf", "KO", package = "faahKO"), 4),
                    rep(system.file("cdf", "WT", package = "faahKO"), 4))
```

Before performing the feature grouping we inspect the result object. With
`featureDefinitions` we can extract the results from the correspondence
analysis.

```{r fdev}
featureDefinitions(xdata)
```

Each row in this data frame represents the definition of one feature, with its
average and range of m/z and retention time. Column `"peakidx"` provides the
index of each chromatographic peak which is assigned to the feature in the
`chromPeaks` matrix of the result object. The `featureValues` function allows to
extract *feature values*, i.e. a matrix with feature abundances, one row per
feature and columns representing the samples of the present data set.

Below we extract the feature values with and without *filled-in* peak
data. Without the gap-filled data only abundances from **detected**
chromatographic peaks are reported. In the gap-filled data, for samples in which
no chromatographic peak for a feature was detected, all signal from the m/z -
retention time range defined based on the detected chromatographic peaks was
integrated.

```{r filled-not-filled}
head(featureValues(xdata, filled = FALSE))
head(featureValues(xdata, filled = TRUE))
```

In total `r nrow(featureDefinitions(xdata))` features have been defined in the
present data set, many of which most likely represent signal from different ions
(adducts or isotopes) of the same compound. The aim of the grouping functions of
are now to define which features most likely come from the same original
compound. The feature grouping functions base on the following
assumptions/properties of LC-MS data:

- Features (ions) of the same compound should have similar retention time.
- The abundance of features (ions) of the same compound should have a similar
  pattern across samples, i.e. if a compound is highly concentrated in one
  sample and low in another, all ions from it should follow the same pattern.
- The peak shape of extracted ion chromatograms (EIC) of features of the same
  compound should be similar as it should follow the elution pattern of the
  original compound from the LC.

The main method to perform the feature grouping is called `groupFeatures` which
takes an `XCMSnExp` object (result object from the `xcms` pre-processing) as
input as well as a parameter object to chose the grouping algorithm and specify
its settings. `xcms` provides and supports the following grouping approaches:

- `SimilarRtimeParam`: perform an initial grouping based on similar retention
  time.
- `AbundanceSimilarityParam`: perform a feature grouping based on correlation
  of feature abundances (values) across samples.
- `EicSimilarityParam`: perform a feature grouping based on correlation of
  EICs.

Calling `groupFeatures` on an `xcms` result object will perform a feature
grouping assigning each feature in the data set to a *feature group*. These
feature groups are stored as an additional column called `"feature_group"` in
the `featureDefinition` data frame of the result object and can be accessed with
the `featureGroups` function. Any subsequent `groupFeature` call will
*sub-group* (refine) the identified feature groups further. It is thus possible
to use a single grouping approach, or to combine multiple of them to generate
the desired feature grouping. While the individual feature grouping algorithms
can be called in any order, it is advisable to use the `EicSimilarityParam` as
last refinement step, because it is the computationally most expensive one,
especially if applied to a result object without any pre-defined feature groups
or if the feature groups are very large. In the subsequent sections we will
apply the various feature grouping approaches subsequently.

Note also that we perform here a grouping of all defined features, but it would
also be possible to *just* group a subset of interesting features (e.g. features
found significant by a statistical analysis of the data set). This is described
in the last section of this vignette.


## Grouping of features by similar retention time

The most intuitive and simple way to group features is based on their retention
time. Before we perform this initial grouping we evaluate retention times and
m/z of all features in the present data set.

```{r feature-rt-mz-plot, fig.width = 8, fig.height = 6, fig.cap = "Plot of retention times and m/z for all features in the data set."}
plot(featureDefinitions(xdata)$rtmed, featureDefinitions(xdata)$mzmed,
     xlab = "retention time", ylab = "m/z", main = "features",
     col = "#00000080")
grid()
```

Several features with about the same retention time (but different m/z) can be
seen, especially at the beginning of the LC. We thus below group features within
a retention time window of 10 seconds into *feature groups*.

```{r}
xdata <- groupFeatures(xdata, param = SimilarRtimeParam(10))
```

The results from the feature grouping can be accessed with the `featureGroups`
function. Below we determine the size of each of these feature groups (i.e. how
many features are grouped together).

```{r}
table(featureGroups(xdata))
```

In addition we visualize these feature groups with the `plotFeatureGroups`
function which shows all features in the m/z - retention time space with grouped
features being connected with a line.

```{r feature-groups-rtime-plot, fig.width = 8, fig.height = 6, fig.cap = "Feature groups defined with a rt window of 10 seconds"}
plotFeatureGroups(xdata)
grid()
```

Let's assume we don't agree with this feature grouping, also knowing that there
were quite large shifts in retention times between runs. We thus re-perform the
feature grouping based on similar retention time with a larger rt window. Prior
to the `groupFeatures` call we have however to drop the previously defined
feature groups as otherwise these would be simply *refined* (i.e. further
subgrouped).

```{r repeat}
## Remove previous feature grouping results to repeat the rtime-based
## feature grouping with different setting
featureDefinitions(xdata)$feature_group <- NULL

## Repeat the grouping
xdata <- groupFeatures(xdata, SimilarRtimeParam(20))
table(featureGroups(xdata))
```

```{r feature-groups-rtime-plot2, fig.width = 8, fig.height = 6, fig.cap = "Feature groups defined with a rt window of 20 seconds"}
plotFeatureGroups(xdata)
grid()
```

Grouping by similar retention time grouped the in total
`r nrow(featureDefinitions(xdata))` features into
`r length(unique(featureGroups(xdata)))` feature groups.


## Grouping of features by abundance correlation across samples

Assuming we are OK with the *crude* initial feature grouping from the previous
section, we can next *refine* the feature groups considering also the feature
abundances across samples. We can use the `groupFeatures` method with an
`AbundanceSimilarityParam` object. This approach performs a pairwise
correlation between the feature values (abundances; across samples) between all
features of a predefined feature group (such as defined in the previous
section). Features that have a correlation `>= threshold` are grouped
together. Feature grouping based on this approach works best for features with a
higher variability in their concentration across samples. Parameter `subset`
allows to restrict the analysis to a subset of samples and allows thus to
e.g. exclude QC sample pools from this correlation as these could artificially
increase the correlation. Other parameters are passed directly to the internal
`featureValues` call that extracts the feature values on which the correlation
should be performed.

Before performing the grouping we could also evaluate the correlation of
features based on their (log2 transformed) abundances across samples with a
heatmap.

```{r abundance-correlation-heatmap, fig.cap = "Correlation of features based on feature abundances.", fig.width = 6, fig.height = 16}
library(pheatmap)
fvals <- log2(featureValues(xdata, filled = TRUE))

cormat <- cor(t(fvals), use = "pairwise.complete.obs")
ann <- data.frame(fgroup = featureGroups(xdata))
rownames(ann) <- rownames(cormat)

res <- pheatmap(cormat, annotation_row = ann, cluster_rows = TRUE,
                cluster_cols = TRUE)
```

Some large correlations can be observed for several groups of features, but many
of them are not within the same *feature group* that were defined in the
previous section (i.e. are not eluting at the same time).

Below we use the `groupFeatures` with the `AbundanceSimilarityParam` to group
features with a correlation higher than 0.7 including both detected and
filled-in signal. Whether filled-in or only detected signal should be used in
the correlation analysis should be evaluated from data set to data set. By
specifying `transform = log2` we tell the function to log2 transform the
abundance prior to the correlation analysis. See the help page for
`groupFeatures` with `AbundanceSimilarityParam` in the `xcms` package for
details and options.

```{r abundance-correlation}
xdata <- groupFeatures(xdata, AbundanceSimilarityParam(threshold = 0.7,
                                                       transform = log2),
                       filled = TRUE)
table(featureGroups(xdata))
```

Many of the larger retention time-based feature groups have been splitted into
two or more sub-groups based on the correlation of their feature abundances. We
evaluate this for one specific feature group `"FG.040"` by plotting their
pairwise correlation.

```{r abundance-correlation-fg040, fig.width = 8, fig.height = 8, fig.cap = "Pairwise correlation plot for all features initially grouped into the feature group FG.040."}
fts <- grep("FG.040", featureGroups(xdata))
pairs(t(fvals[fts, ]), gap = 0.1, main = "FG.040")
```

Indeed, correlations can be seen only between some of the features in this
retention time feature group, e.g. between *FT117* and *FT120* and between
*FT195* and *FT200*. Note however that this abundance correlation suffers from
relatively few samples (8 in total), and a relatively small variance in
abundances across these samples.

After feature grouping by abundance correlation, the
`r nrow(featureDefinitions(xdata))` features have been grouped into
`r length(unique(featureGroups(xdata)))` feature groups.


## Grouping of features by similarity of their EICs

The chromatographic peak shape of an ion of a compound should be highly similar
to the elution pattern of this compound. Thus, features from the same compound
are assumed to have similar peak shapes of their EICs **within the same
sample**. A grouping of features based on similarity of their EICs can be
performed with the `groupFeatures` and the `EicSimilarityParam` object.  It is
advisable to perform the peak shape correlation only on a subset of samples
(because peak shape correlation is computationally intense and because
chromatographic peaks of low intensity features are notoriously noisy).  The
`EicSimilarityParam` approach has thus the parameter `n` which allows to select
the number of top samples (ordered by total intensity of feature abundances per
feature group) on which the correlation should be performed. With an value of `n
= 3`, the 3 samples with the highest signal for all features in that group will
be first identified for each feature group and then a pairwise similarity
calculation will be performed within each of these samples. The resulting
similarity score from these 3 samples will then be aggregated into a single
score by taking the 75% quantile across the 3 samples. This value is then
subsequently compared with the cut-off for similarity (parameter `threshold`)
and features with a score `>= threshold` are grouped into the same feature
group.

Below we group the features based on similarity of their EICs in the two samples
with the highest total signal for the respective feature groups. By default, a
Pearson correlation coefficient is used as similarity score but any
similarity/distance metric function could be used instead (parameter `FUN` of
the `EicSimilarityParam` - see the respective help page `?EicSimilarityParam`
for details and options). We define as a threshold a correlation coefficient of
0.7.

```{r correlate-eic, message = FALSE}
xdata <- groupFeatures(xdata, EicSimilarityParam(threshold = 0.7, n = 2))
```

This is the most computationally intense approach since it involves also loading
the raw MS data to extract the ion chromatograms for each feature. The results
of the grouping are shown below.

```{r correlate-eic-result}
table(featureGroups(xdata))
```

In most cases, pre-defined feature groups (by the abundance correlation) were
not further subdivided. Below we evaluate some of the feature groups, starting
with *FG.008.001* which was split into two different feature groups based on EIC
correlation. We first extract the EICs for all features from this initial
feature group. With `n = 1` we specify to extract the EIC only from the sample
with the highest intensity.

```{r}
fts <- grep("FG.008.001", featureGroups(xdata))
eics <- featureChromatograms(xdata, features = fts,
                             filled = TRUE, n = 1)
```

Next we plot the EICs using a different color for each of the subgroups. With
`peakType = "none"` we disable the highlighting of the detected chromatographic
peaks.

```{r example-1-eic, fig.width = 8, fig.height = 6, fig.cap = "EICs of features from feature group FG.008.001 in the same sample. Shown are the actual intensities (left) and intensities normalized to 1 (right). Features being part of the same feature group after grouping by EIC similarity are shown in the same color."}
cols <- c("#ff000080", "#00ff0080")
names(cols) <- unique(featureGroups(xdata)[fts])

par(mfrow = c(1, 2))
plotChromatogramsOverlay(eics, col = cols[featureGroups(xdata)[fts]],
                         lwd = 2, peakType = "none")
plotChromatogramsOverlay(normalize(eics),
                         col = cols[featureGroups(xdata)[fts]],
                         lwd = 2, peakType = "none")
```

One of the features within the original feature group was separated from the
other two because of a low similarity of their EICs. In fact, the feature's EIC
is shifted in retention time dimension and can thus not represent the signal
from the same compound.

We evaluate next the sub-grouping in another feature group.

```{r}
fts <- grep("FG.068.001", featureGroups(xdata))
eics <- featureChromatograms(xdata, features = fts,
                             filled = TRUE, n = 1)
```

Next we plot the EICs using a different color for each of the subgroups.

```{r example-2-eic, fig.width = 8, fig.height = 6, fig.cap = "EICs for features from feature group FG.068.001 in the same sample. Shown are the actual intensities (left) and intensities normalized to 1 (right). Features being part of the same feature group after grouping by EIC similarity are shown in the same color."}
cols <- c("#ff000080", "#00ff0080")
names(cols) <- unique(featureGroups(xdata)[fts])

par(mfrow = c(1, 2))
plotChromatogramsOverlay(eics, col = cols[featureGroups(xdata)[fts]],
                         lwd = 2, peakType = "none")
plotChromatogramsOverlay(normalize(eics),
                         col = cols[featureGroups(xdata)[fts]],
                         lwd = 2, peakType = "none")

```

Based on the EIC correlation, the initial feature group *FG.068.001* was grouped
into two separate sub-groups.

The grouping based on EIC correlation on the pre-defined feature groups from the
previous sections grouped the `r nrow(featureDefinitions(xdata))` features into
`r length(unique(featureGroups(xdata)))` feature groups.

## Grouping of subsets of features

In the previous sections we were always considering all features from the data
set, but sometimes it could be desirable to just group a pre-defined set of
features, for example features found to be of particular interest in a certain
experiment (e.g. significant features). This can be easily achieved by assigning
the features of interest to a initial feature group, using `NA` as group ID
for all other features.

To illustrate this we *reset* all feature groups by setting them to `NA` and
assign our features of interest (in this example just 30 randomly selected
features) to an initial feature group `"FG"`.

```{r reset-feature-groups}
featureDefinitions(xdata)$feature_group <- NA_character_

set.seed(123)
fts_idx <- sample(1:nrow(featureDefinitions(xdata)), 30)
featureDefinitions(xdata)$feature_group[fts_idx] <- "FG"
```

Any call to `groupFeatures` would now simply sub-group this set of 30
features. Any feature which has an `NA` in the `"feature_group"` column will be
ignored.

```{r rtime-grouping}
xdata <- groupFeatures(xdata, SimilarRtimeParam(diffRt = 20))
xdata <- groupFeatures(xdata, AbundanceSimilarityParam(threshold = 0.7))
table(featureGroups(xdata))
```


# Session information

```{r sessionInfo}
sessionInfo()
```

# References