---
title: "Managing Mass Spectrometry Experiments"
output:
    BiocStyle::html_document:
        toc_float: true
vignette: >
    %\VignetteIndexEntry{Managing Mass Spectrometry Experiments}
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding{UTF-8}
    %\VignettePackage{MsExperiment}
    %\VignetteDepends{BiocStyle,rpx,Spectra}
---

```{r style, echo = FALSE, results = 'asis', message=FALSE}
BiocStyle::markdown()
```

**Package**: `r BiocStyle::Biocpkg("MsExperiment")`<br />
**Authors**: `r packageDescription("MsExperiment")[["Author"]] `<br />
**Last modified:** `r file.info("MsExperiment.Rmd")$mtime`<br />
**Compiled**: `r date()`

```{r, echo = FALSE, message = FALSE}
library(MsExperiment)
library(Spectra)
library(BiocStyle)
```

# Introduction

The goal of the `r Biocpkg("MsExperiment")` package is to provide a container
for all data related to a mass spectrometry (MS) experiment. Also other
Bioconductor packages allow to represent MS experiment data (such as the
`r Biocpkg("MSnbase")` package). The `MsExperiment` however aims at being very
light-weight and flexible to accommodate all possible types of MS experiments
(proteomics, metabolomics, ...) and all types of MS data representations
(chromatographic and spectral data, quantified features etc). In addition, it
allows to bundle additional files and data, such as annotations, within the
object.

In this vignette, we will describe how to create a `MsExperiment` object and
populate it with various types of data.

```{r load_pkg, message = FALSE}
library("MsExperiment")
```

We will also use the `r Biocpkg("Spectra")` package to import MS data and thus
load it here too.

```{r load_spectra, message = FALSE}
library("Spectra")
```


# Installation

The package can be installed with the `r Biocpkg("BiocManager")` package. To
install `BiocManager` use `install.packages("BiocManager")` and, after that,
`BiocManager::install("MsExperiment")` to install `r Biocpkg("MsExperiment")`
which will install the package including all required dependencies.


# Getting data

We will use a small subset of the
[PXD022816](https://www.ebi.ac.uk/pride/archive/projects/PXD022816)
project ([Morgenstern et
al. (2020)](https://doi.org/10.1021/acs.jproteome.0c00956)). The
acquisitions correspond to a Pierce Thermo HeLa digestion standard,
diluted to 50ng/uL with 97:3 + 0.1% formic acid, and acquired on a
QExactive instrument.

Below, we use the `r Biocpkg("rpx")` package to access the project
from the PRIDE repository, and download files of interest. Note that
these will automatically be cached in the `rpx` packages' cache
directory.

```{r px, eval = TRUE}
library("rpx")
px <- PXDataset("PXD022816")
px
pxfiles(px)
```

The project provides the vendor raw files, the converted mzML files as
well as the identification mzid files. Let's download fractions 1 and
2 of the mzML files.

If you run these commands interactively and it's the first time you
use `pxget()`, you will be asked to create the `rpx` cache directory -
you can safelfy answer *yes*. The files will then be downloaded. Next
time you want to get the same files, they will be loaded automatically
from cache.

```{r fls, eval = TRUE}
(i <- grep(".+0[12].+mzML$", pxfiles(px), value = TRUE))
fls <- pxget(px, i)
fls
```

# Mass spectrometry experiment

Let's start by creating an empty `MsExperiment` object that we will
populate with different pieces of data as we proceed with the analysis
of our data.

```{r make_exp, eval = TRUE}
msexp <- MsExperiment()
msexp
```

## Experiment files

Let's now start with our MS experiment management by saving the
relevant files in a dedicated `MsExperimentFiles` object. In addition
to the mzML files, let's also assume we have the human proteomics
fasta file ready. Later, when loading the raw data into R, we will
refer directly to the files in this `MsExperimentFiles` object.

```{r make_exp_fls, eval = TRUE}
msfls <- MsExperimentFiles(mzmls = fls,
                           fasta = "homo_sapiens.fasta")
msfls
```

Let's add these files to the main experiment management object:

```{r add_exp_fls, eval = TRUE}
experimentFiles(msexp) <- msfls
msexp
```

## Experimental design

The `sampleData` slot is used to describe the overall experimental design of the
experiment. It can be used to specify the samples of the experiment and to
relate them to the files that are part of the experiment. There can be a
one-to-one link between a sample and a file, such as for example in label-free
approaches, or one-to-many, in labelled multiplexed approaches.

Here, we create a simple data frame with sample annotations that include the
original file names and the respective fractions.

```{r add_sample_data, eval = TRUE}
sampleData(msexp) <- DataFrame(
    mzmls = basename(experimentFiles(msexp)[["mzmls"]]),
    fractions = 1:2)
sampleData(msexp)
```

## Raw data

We can now create a `Spectra` object containing the raw data stored in
the mzML files. If you are not familiar with the `Spectra` object,
please refer to the [package
vignettes](https://rformassspectrometry.github.io/Spectra/articles/Spectra.html).

```{r sp, eval = TRUE}
sp <- Spectra(experimentFiles(msexp)[["mzmls"]])
sp
```

We can now add this object to the main experiment management object:

```{r add_sp, eval = TRUE}
spectra(msexp) <- sp
msexp
```

## Third party applications

Let's now assume we want to search the spectra in our mzML files
against the `homo_sapiens.fasta` file. To do so, we would like to use
a search engine such as MSGF+, that is run using the command line and
generates mzid files.

The command to run MSGF+ would look like this (see the [manual
page](https://msgfplus.github.io/msgfplus/MSGFPlus.html) for details):

```
java -jar /path/to/MSGFPlus.jar \
     -s input.mzML \
     -o output.mzid
     -d proteins.fasta \
     -t 20ppm \ ## precursor mass tolerance
     -tda 1 \   ## search decoy database
     -m 0 \     ## fragmentation method as written in the spectrum or CID if no info
     -int 1     ## Orbitrap/FTICR/Lumos
```

We can easily build such a command for each of our input file:

```{r make_cmd, eval = TRUE}
mzids <- sub("mzML", "mzid", basename(experimentFiles(msexp)[["mzmls"]]))
paste0("java -jar /path/to/MSGFPlus.jar",
       " -s ", experimentFiles(msexp)[["mzmls"]],
       " -o ", mzids,
       " -d ", experimentFiles(msexp)[["fasta"]],
       " -t 20ppm",
       " -m 0",
       " int 1")
```

Here, for the sake of time and portability, we will not actually run
MSGF+, but a simple shell script that will generate mzid files in a
temporary R directory.


```{r touch, eval = TRUE}
(output <- file.path(tempdir(), mzids))
cmd <- paste("touch", output)
cmd
```

The `cmd` variable holds the two commands to be run on the command
line that will generate the new files. We can run each of these
commands with the `system()` function.

```{r system, eval = TRUE}
sapply(cmd, system)
```

Below, we add the names of the newly created files to our experiment:

```{r add_mzids, eval = TRUE}
experimentFiles(msexp)[["mzids"]] <- mzids
experimentFiles(msexp)
msexp
```

We can also decide to store the commands that were used to generate
the mzid files in the experiment's metadata slot. Here, we use the
convention to name that metadata item `"mzmls_to_mzids"` to document
to input and output of these commands.

```{r add_cmd, eval = TRUE}
metadata(msexp)[["mzmls_to_mzids"]] <- cmd
metadata(msexp)
```

Finally, the `existMsExperimentFiles()` can be used at any time to
check which of files that are associated with an experiment actually
exist:

```{r exp_exists, eval = TRUE}
existMsExperimentFiles(msexp)
```

# Saving and reusing experiments

The `MsExperiment` object has been used to store files and data
pertaining to a mass spectrometry experiment. It is now possible to
save that object and reload it later to recover all data and metadata.
See also section *Using `MsExperiment` with `MsBackendSql`* below for an
alternative to load/restore defined MS experiments from an SQL database.

```{r save_exp, eval = TRUE}
saveRDS(msexp, "msexp.rds")
rm(list = ls())
```

```{r read_exp, eval = TRUE}
msexp <- readRDS("msexp.rds")
msexp
experimentFiles(msexp)
```

We can access the raw data as long as the mzML files that were used to
generate it still exist in their original location, which is the case
here as they were saved in the `rpx` cache directory.

```{r plot_sp, eval = TRUE}
sp <- spectra(msexp)
sp
plotSpectra(sp[1000])
```


# Linking experimental data to samples

For some experiments and data analyses an explicit link between data, data files
and respective samples is required. Such links enable an easy (and error-free)
subset or re-ordering of a whole experiment by sample and would also
simplify coloring and labeling of the data depending on the sample or of its
variables or conditions.

Below we generate an `MsExperiment` object for a simple experiment consisting of
a single sample measured in two different injections to the same LC-MS setup.

```{r make_exp2}
lmse <- MsExperiment()
sd <- DataFrame(sample_id = c("QC1", "QC2"),
                sample_name = c("QC Pool", "QC Pool"),
                injection_idx = c(1, 3))
sampleData(lmse) <- sd
```

We next add mzML files to the experiment for the sample that was measured. These
are available within the `msdata` R package. We add also an additional
*annotation* file `"internal_standards.txt"` to the experiment, which could be
e.g. a file with m/z and retention times of internal standards added to the
sample (note that such files don't necessarily have to exist).

```{r add_fls2}
fls <- dir(system.file("sciex", package = "msdata"), full.names = TRUE)
basename(fls)

experimentFiles(lmse) <- MsExperimentFiles(
    mzML_files = fls,
    annotations = "internal_standards.txt")
```

Next we load the MS data from the mzML files as a `Spectra` object and add them
to the experiment (see the vignette of the `r BiocStyle::Biocpkg("Spectra")` for
details on import and representation of MS data).

```{r add_sp2}
sps <- Spectra(fls, backend = MsBackendMzR())
spectra(lmse) <- sps
lmse
```

At this stage we have thus sample annotations and MS data in our object, but no
explicit relationships between them. Without such linking between files and
samples any subsetting would only subset the `sampleData` but not any of the
potentially associated files. We next use the `linkSpectraData` function
to establish and define such relationships. First we link the experimental files
to the samples: we want to link the first mzML file in the element called
`"mzML_file"` in the object's `experimentFiles` to the first row in `sampleData`
and the second file to the second row.

```{r link_sample_data}
lmse <- linkSampleData(lmse, with = "experimentFiles.mzML_file",
                        sampleIndex = c(1, 2), withIndex = c(1, 2))
```

To define the link we have thus to specify with which *element* within our
`MsExperiment` we want to link samples. This can be done with the parameter
`with` that takes a single `character` representing the name (*address*) of the
data element. The name is a combination of the name of the slot within the
`MsExperiment` and the name of the element (or column) within that slot
separated by a `"."`. Using `with = "experimentFiles.mzML_file"` means we want
to link samples to values within the `"mzML_file"` element of the object's
`experimentFiles` slot - in other words, we want to link samples to values in
`experimentFiles(lmse)$mzML_file`. The indices of the rows (samples) in
`sampleData` and the indices of the values in `with` to which we want to link
the samples can be defined with `sampleIndex` and `withIndex`. In the example
above we used `sampleIndex = c(1, 2)` and `withIndex = c(1, 2)`, thus, we want
to link the first row in `sampleData` to the first value in `with` and the
second row to the second value. See also the section *Linking sample data to
other experimental data* in the documentation of `MsExperiment` for more
information and details.

What happened internally by the call above is illustrated in the figure
below. The link is represented as a two-column integer `matrix` with the indices
of the linked sample in the first and the indices of the associated elements in
the second columns (this matrix is essentially a
`cbind(sampleIndex, withIndex)`).

```{r, echo = FALSE}
knitr::include_graphics("imgs/Links_01.png")
```

We next establish a second link between each sample and the *annotation*
file `"internal_standards.txt"` in `experimentFiles(lmse)$standards`:

```{r link_sample_data2}
lmse <- linkSampleData(lmse, with = "experimentFiles.annotations",
                        sampleIndex = c(1, 2), withIndex = c(1, 1))
```

The figure below illustrates again what happened internally by this call: a new
*link matrix* was added establishing the relationship between the two samples
and the one value in `experimentFiles(lmse)$annotations`.

```{r, echo = FALSE}
knitr::include_graphics("imgs/Links_02.png")
```

It is thus also possible to link different samples to the same element. We next
link the spectra in the object to the individual samples. We use for that an
alternative way to specify the link without the need to provide `sampleIndex`
and `withIndex`. Sample-to-data links can also be specified using a syntax
similar to an SQL join:
`"sampleData.<column in sampleData> = <slot>.<element in slot>"`. Links will be
thus established between elements with matching values in the specified data
fields (i.e. between rows in `sampleData` for which values in the specified
column matches values in `<slot>.<element>`). In order to use this alternative
approach to link spectra to the respective samples we have to first add the
(full) raw file name as an additional column to the object's `sampleData`. We
can now add links between spectra and samples by matching this raw file name to
the original file name from which the spectra were imported (which is available
in the `"dataOrigin"` spectra variable).

```{r link_sample_data3}
sampleData(lmse)$raw_file <- normalizePath(fls)
lmse <- linkSampleData(
    lmse, with = "sampleData.raw_file = spectra.dataOrigin")
```

The link was thus established between matching values in
`sampleData(lmse)$raw_file` and `spectra(lmse)$dataOrigin`.

```{r show_link}
sampleData(lmse)$raw_file
spectra(lmse)$dataOrigin |>
                head()
```

The figure below illustrates this link. With that last call we have thus
established links between samples and 3 different data elements in
the `MsExperiment`.

```{r, echo = FALSE}
knitr::include_graphics("imgs/Links_03.png")
```

```{r show_exp2}
lmse
```

A convenience function to quickly extract the index of a sample a spectrum is
associated with is `spectraSampleIndex()`. This function returns an `integer`
vector of length equal to the number of spectra in the object with the row in
the object's `sampleData` a spectrum is linked to, or `NA_integer_` if a
spectrum is not linked to any sample.

```{r}
#' Show the sample assignment for the first few spectra
spectraSampleIndex(lmse) |>
    head()
```

If we had also quantified *feature* values, we could also link them to the
samples. Below we create a simple, small `SummarizedExperiment` to represent
such quantified feature values and add that to our experiment. To show that
`MsExperiment` supports also links between subsets of data elements, we create a
`SummarizedExperiment` that contains values for an additional sample which is
not present in our `sampleData`. Also, we add samples in an arbitrary order.

```{r add_se, message = FALSE}
library(SummarizedExperiment)
sd <- DataFrame(sample = c("QC2", "QC1", "QC3"), idx = c(3, 1, 5))
se <- SummarizedExperiment(colData = sd, assay = cbind(1:10, 11:20, 21:30))

qdata(lmse) <- se
```

Next we link the samples in this `SummarizedExperiment` to the samples to in the
`MsExperiment` using matching values between the `"sample_id"` column in the
object's `sampleData` data frame and the column `"sample"` in the
`SummarizedExperiment`'s `colData` which is stored in the `@qdata` slot. The
naming convention to define such columns is `<slot name>.<column name>`.

```{r link_sample_data4}
sampleData(lmse)$sample_id
qdata(lmse)$sample

lmse <- linkSampleData(lmse, with = "sampleData.sample_id = qdata.sample")
lmse
```

The main advantage of all these links is that any subsetting of the experiment
by sample will keep the (linked) data consistent. To illustrate this we subset
below the experiment to the second sample.

```{r subset_exp2}
b <- lmse[2]
b
```

The subset object contains now all data elements that are linked to this
second sample. Accessing the `assay` of the `SummarizedExperiment` in
`qdata` will thus return only the quantified feature abundances for this
second sample.

```{r extract_exp2}
assay(qdata(b))
```

But what happens for data elements that are not linked to any sample? Below we
add a `data.frame` as a `metadata` to the experiment and subset the object
again.

```{r add_metadata}
metadata(lmse)$other <- data.frame(
                  sample_name = c("study_1", "POOL", "study_2"),
                  index = 1:3)
b <- lmse[2]
metadata(b)
```

By default, any element which is **not** linked to a sample is retained in the
filtered/subset object.

We next link each sample to the second row in this data frame and subset the
data again to the second sample.

```{r link_sample_data5}
lmse <- linkSampleData(lmse, with = "metadata.other",
                      sampleIndex = 1:2, withIndex = c(2, 2))
b <- lmse[2]
metadata(b)
```

Subsetting thus retained only the row in the data frame for the linked
sample. Obviously it is also possible to subset to multiple samples, in
arbitrary order. Below we re-order our experiment.

```{r show_subset_exp2}
lmse <- lmse[c(2, 1)]
sampleData(lmse)
```

The sample order is thus reversed and also all other linked elements are
re-ordered accordingly, such as `"mzML_file"` in the object's `experimentFiles`.

```{r show_expfls2_msml}
experimentFiles(lmse)$mzML_file
```

It is however important to note, that subsetting will also *duplicate* elements
that are associated with multiple samples:

```{r show_explfs2_annot}
experimentFiles(lmse)$annotations
```

Thus, while we added a single *annotation* file to the data element
`"annotations"` in `experimentFiles`, after subsetting we ended up with two
identical files. This duplication of *n:m* relationships between
samples to elements does however not affect data consistency. A sample will
always be linked to the correct value/element.


# Subset and filter `MsExperiment`

As already shown above, `MsExperiment` objects can be subset with `[` which will
subset the data by sample. Depending on whether relationships (links) between
samples and any other data within the object are present also these are
correctly subset. In addition to this general subset operation, it is possible
to individually filter the spectra data within an `MsExperiment` using the
`filterSpectra` function. This function takes any filter function supported by
`Spectra` with parameter `filter`. Parameters for this filter function can be
passed through `...`. As an example we filter below the spectra data of our
`MsExperiment` keeping only spectra with an retention time between 200 and 210
seconds.

```{r}
#' Filter the Spectra using the `filterRt` function providing also the
#' parameters for this function.
res <- filterSpectra(lmse, filterRt, rt = c(200, 210))
res
```

The resulting `MsExperiment` contains now much fewer spectra. `filterSpectra`
did only filter the spectra data, but not any of the other data slots. It did
however update and consolidate the relationships between samples and spectra
(if present) after filtering:

```{r}
#' Extract spectra of the second sample after filtering
spectra(res[2L])
```


# Using `MsExperiment` with `MsBackendSql`

The `r Biocpkg("MsBackendSql")` provides functionality to store mass
spectrometry data in a SQL database and to *represent* such data as a `Spectra`
object (through its `MsBackendSql` or `MsBackendOfflineSql` backends). Next to
the MS data, it is also possible to store sample data in such SQL databases
hence allowing to create self-contained databases for MS experiments. This
simplifies the process to load pre-defined MS experiment or to share experiment
data across analyses or with a collaborator. In this section we show how MS data
and sample information from an experiment can be stored into a SQL database and
how that data can then be loaded again as a `MsExperiment`.

Below we first define the raw data files of the experiment. In our example we
use two small test files provided with the `r Biocpkg("msdata")` package.

```{r}
fls <- c(system.file("microtofq", "MM14.mzML", package = "msdata"),
         system.file("microtofq", "MM8.mzML", package = "msdata"))
```

We can then use the `createMsBackendSqlDatabase` function from the
`r Biocpkg("MsBackendSql")` package to write the data into a SQLite
database. SQLite databases are particularly easy to create, use and also to
share, but for large experiments it is suggested to use more powerful database
engines, such as for example *MariaDB* (see also the documentation and vignette
of the `r Biocpkg("MsBackendSql")` package for parameters and settings for such
large databases).

Also, in our example we store the SQLite database into a data temporary file,
but for a real use case we would want to use a *real* file.

```{r}
library(MsBackendSql)
library(RSQLite)

#' Create the SQLite database where to store the data. For our
#' example we create the database in a temporary file.
sql_file <- tempfile()
con <- dbConnect(SQLite(), dbname = sql_file)

#' Write the MS data to the.
createMsBackendSqlDatabase(dbcon = con, fls)
```

We can now create a `Spectra` object representing the MS data in that database
using either a `MsBackendSql` or `MsBackendOfflineSql` backend.

```{r}
#' Load the database as a Spectra object
sps <- Spectra(sql_file, source = MsBackendOfflineSql(), drv = SQLite())
sps
```

Next we define sample annotations for the two files and create a `MsExperiment`
with the MS data and the related sample annotations.

```{r}
#' Define sample annotations. Ideally all experiment-relavant information
#' should be specified.
sdata <- data.frame(file_name = basename(fls),
                    sample_name = c("MM14", "MM8"),
                    sample_group = c("ctrl", "ctrl"))

#' To simplify subsequent linking between samples and data files we
#' define a new spectra variable with the file names of the raw data files.
sps$file_name <- basename(dataOrigin(sps))

#' Create the MsExperiment for that data
mse <- MsExperiment(sampleData = sdata, spectra = sps)
```

Finally we *link* the MS spectra to the individual samples using the file names
of the original data files.

```{r}
#' Establish the mapping between spectra and samples
mse <- linkSampleData(mse, with = "sampleData.file_name = spectra.file_name")
mse
```

With sample annotations and the mapping between samples and spectra defined, we
can next write this information to the SQL database containing already the MS
data of our experiment: the `dbWriteSampleData` function writes, for
`MsExperiment` objects that use a `Spectra` with a `MsBackendSql` (or
`MsBackendOfflineSql`) backend the available sample data to additional database
tables in the database.

```{r}
#' Write sample data (and mapping between samples and spectra) to the
#' database
dbWriteSampleData(mse)
```

The SQL database does now contain the MS data, the sample annotations and the
mapping between samples and spectra. Creation of such a database needs to be
performed only once for an experiment. The associated SQLite database file could
now be shared with a collaborator or used across different analyses without the
need to share or copy also the raw data files or sample annotations (which would
usually needed to be provided as a separate file).

To load the MS experiment data again into R, we first need to create a `Spectra`
object with a `MsBackendSql` or `MsBackendOfflineSql` backend for that SQL
database.

```{r}
#' Create a Spectra object for the MS data of that database
sps <- Spectra(sql_file, source = MsBackendOfflineSql(), drv = SQLite())
```

We then pass this `Spectra` object with parameter `spectra` to the
`MsExperiment` constructor call. The function will then read also sample data
from the database (if available) as well as the mapping between samples and
spectra to restore our MS experiment.

```{r}
#' Create the MsExperiment reading MS data and sample data from
#' the database
mse <- MsExperiment(spectra = sps)
mse
```

We have thus now the full MS experiment data available.

For smaller experiments, such SQL databases could also be created with an
alternative, eventually simpler, approach. For that we first use the
`readMsExperiment` function to create an `MsExperiment` from the file names of
the raw MS data files and a `data.frame` with the associated sample
annotations. We re-use here the variables `fls` with the data file names and
`sdata` with the sample data.

```{r}
#' Create an MsExperiment with data from two (raw) data files
#' and associated sample annotations.
mse <- readMsExperiment(spectraFiles = fls, sampleData = sdata)
mse
```

The MS data in that `MsExperiment` is represented by a `Spectra` object using
the `MsBackendMzR`:

```{r}
#' Show the Spectra from the experiment
spectra(mse)
```

We next change the backend of this `Spectra` object using the `setBackend`
function to a `MsBackendOfflineSql`. We choose again a SQLite database format
and store the data to a temporary file.

```{r}
#' Define the SQLite database file
sql_file2 <- tempfile()

#' Change the backend of the Spectra data
spectra(mse) <- setBackend(spectra(mse), MsBackendOfflineSql(),
                           dbname = sql_file2, drv = SQLite())

spectra(mse)
```

All MS data is now stored in the SQLite database and the `Spectra` object of our
experiment uses the `MsBackendOfflineSql` to represent/interface this data. To
store also the sample data to the database we can use the `dbWriteSampleData` as
before.

```{r}
#' Store also sample data to the database
dbWriteSampleData(mse)
```

In addition to create self-contained SQL databases for an experiment, this code
could also be used within an analysis workflow to serialize/de-serialize
results, always with the advantage of keeping the full experiment data in a
single place (file) in a language-agnostic format.

We can for example use basic functions from the *DBI*/*RSQLite* packages (or
simple SQL commands) to access the data in the database. Below we connect to the
database and list the available tables.

```{r}
#' Connect to the database
con <- dbConnect(SQLite(), dbname = sql_file2)

#' List the available database tables
dbListTables(con)
```

The *msms_spectrum* table contains general metadata for the individual MS
spectra, *msms_spectrum_peak_blob* the actual peak data (*m/z* and intensity
values), *sample_data* the sample annotations and *sample_to_msms_spectrum* the
link between samples and spectra. Below we retrieve the content of the
*sample_data* table.

```{r}
#' Get the content of the sample_data table
dbGetQuery(con, "select * from sample_data")

#' Close the connection to the database
dbDisconnect(con)
```


# Session information

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