synergise2 {synapter} | R Documentation

## Synergise identification and quantitation results

### Description

Performs a complete default analysis on the files defined in `filenames`, creates a complete html report and saves/exports all results as `csv` and `rds` files. See details for a description of the pipeline and `Synapter` for manual execution of individual steps.

### Usage

    synergise2(filenames, master = FALSE, object, outputdir,
      outputfile = paste0("synapter_report_", strftime(Sys.time(),
      "%Y%m%d-%H%M%S"), ".html"), fdr = 0.01, fdrMethod = c("BH",
      "Bonferroni", "qval"), fpr = 0.01, peplen = 7, missedCleavages = 2,
      IisL = FALSE, identppm = 20, quantppm = 20, uniquepep = TRUE,
      span.rt = 0.05, span.int = 0.05, grid.ppm.from = 2,
      grid.ppm.to = 20, grid.ppm.by = 2, grid.nsd.from = 0.5,
      grid.nsd.to = 5, grid.nsd.by = 0.5, grid.imdiffs.from = 0.6,
      grid.imdiffs.to = 1.6, grid.imdiffs.by = 0.2, grid.subset = 1,
      grid.n = 0, grid.param.sel = c("auto", "model", "total", "details"),
      fm.ppm = 25, fm.ident.minIntensity = 70,
      fm.quant.minIntensity = 70, fm.minCommon = 1, fm.minDelta = 1,
      fm.fdr.unique = 0.05, fm.fdr.nonunique = 0.05,
      mergedEMRTs = c("rescue", "copy", "transfer"),
      template = system.file("reports", "synergise2.Rmd", package =
      "synapter"), verbose = interactive())

### Arguments

`filenames` | A named `list` of file names to be load. The names must be `identpeptide`, `quantpeptide`, `quantpep3d` and `fasta` (could be an RDS file created by `link{createUniquePeptideDbRds}`). If fragmentmatching should be used `identfragments` (could be skipped if a master RDS files is used for `identpeptide`) and `quantspectra` have to be given as well. `identpeptide` can be a `csv` final peptide file (from PLGS) or a saved `"MasterPeptides"` data object as created by `makeMaster` if working with master peptide data. To serialise the `"MasterPeptides"` instance, use the `saveRDS` function, and file extenstion `rds`.
`master` | A `logical` indicating if the identification final peptide files are master (see `makeMaster`) or regular files. Default is `FALSE`.
`object` | An instance of class `Synapter` that will be copied, processed and returned. If `filenames` are also provided, the latter and `object`'s `inputFiles` will be checked for equality.
`outputdir` | A `character` with the full path to an existing directory.
`outputfile` | A `character` with the file name for the report.
`fdr` | Peptide false discovery rate. Default is 0.01.
`fdrMethod` | P-value adjustment method. One of `"BH"` (default) for Benjamini and HochBerg (1995), `"Bonferroni"` for Bonferroni's single-step adjusted p-values for strong control of the FWER and `"qval"` from the `qvalue` package. See `Synapter` for references.
`fpr` | Protein false positive rate. Default is 0.01.
`peplen` | Minimum peptide length. Default is 7.
`missedCleavages` | Number of allowed missed cleavages. Default is 2.
`IisL` | If `TRUE` Isoleucin and Leucin are treated as equal. In this case sequences like "ABCI", "ABCL" are removed because they are not unqiue. If `FALSE` (default) "ABCI" and "ABCL" are reported as unique.
`identppm` | Identification mass tolerance (in ppm). Default is 20.
`quantppm` | Quantitation mass tolerance (in ppm). Default is 20.
`uniquepep` | A `logical` is length 1 indicating if only unique peptides in the identification and quantitation peptides as well as unique tryptic peptides as defined in the fasta file. Default is `TRUE`.
`span.rt` | The loess span parameter for retention time correction. Default is 0.05.
`span.int` | The loess span parameter for intensity correction. Default is 0.05.
`grid.ppm.from` | Mass tolerance (ppm) grid starting value. Default is 2.
`grid.ppm.to` | Mass tolerance (ppm) grid ending value. Default is 20.
`grid.ppm.by` | Mass tolerance (ppm) grid step value. Default is 2.
`grid.nsd.from` | Number of retention time stdev grid starting value. Default is 0.5.
`grid.nsd.to` | Number of retention time stdev grid ending value. Default is 5.
`grid.nsd.by` | Number of retention time stdev grid step value. Default is 0.5.
`grid.imdiffs.from` | Ion mobility difference grid starting value. value. Default is 0.6.
`grid.imdiffs.to` | Ion mobility difference grid ending value. Default is 1.6.
`grid.imdiffs.by` | Ion mobility difference grid step value. Default is 0.2.
`grid.subset` | Percentage of features to be used for the grid search. Default is 1.
`grid.n` | Absolute number of features to be used for the grid search. Default is 0, i.e ignored.
`grid.param.sel` | Grid parameter selection method. One of `auto` (default), `details`, `model` or `total`. See `Synapter` for details on these selection methods.
`fm.ppm` | Fragment Matching tolerance: Peaks in a range of `fm.ppm` are considered as identical. Default is 25.
`fm.ident.minIntensity` | Minimal intensity of a Identification fragment to be not filtered prior to Fragment Matching.
`fm.quant.minIntensity` | Minimal intensity of a peaks in a Quantitation spectra to be not filtered prior to Fragment Matching.
`fm.minCommon` | Minimal number of peaks that unique matches need to have in common. Default 1.
`fm.minDelta` | Minimal difference in number of peaks that non-unique matches need to have to be considered as true match. Default 1.
`fm.fdr.unique` | Minimal FDR to select `fm.minCommon` automatically (if both values are given the more restrictive one (that filters more fragments) is used). Default 0.05.
`fm.fdr.nonunique` | Minimal FDR to select `fm.minDelta` automatically (if both values are given the more restrictive one (that filters more fragments) is used). Default 0.05.
`mergedEMRTs` | One of `"rescue"` (default), `"copy"` or `"transfer"`. See the documentation for the `findEMRTs` function in `Synapter` for details.
`template` | A `character` full path to Rmd template.
`verbose` | A `logical` indicating if progress output should be printed to the console. Default is `TRUE`.

### Details

In contrast to `synergise1` `synergise2` extends the default analysis and offers the follwing unique features:

- Performing 3D grid search (M/Z, Retention Time, Ion Mobility) for HDMSE data.

- Applying intensity correction.

- Filtering results by fragment matching.

Data can be input as a `Synapter` object if available or as a list of files (see `filenames`) that will be used to read the data in. The html report and result files will be created in the `outputdir` folder. All other input parameters have default values.

The data processing and analysis pipeline is as follows:

1. If `uniquepep` is set to TRUE (default), only unique proteotypic identification and quantitation peptides are retained.

2. Peptides are filtered for a FDR <= `fdr` (default is 0.01) using the "BH" method (see `fdr` and `fdrMethod` parameters for details).

3. Peptide with a mass tolerance > 20 ppms (see `quantppm` and `identppm`) are filtered out.

4. Peptides with a protein false positive rate (as reported by the PLGS software) > `fpr` are filtered out.

5. Common identification and quantitation peptides are merged and a retention time model is created using the Local Polynomial Regression Fitting (`loess` function for the `stats` package) using a default `span.rt` value of 0.05.

6. A grid search to optimise the width in retention time and mass tolerance (and ion mobility for HDMSE) for EMRTs matching is performed. The default grid search space is from 0.5 to 5 by 0.5 retention time model standard deviations (see `grid.nsd.from`, `grid.nsd.to` and `grid.nsd.by` parameters) and from 2 to 20 by 2 parts per million (ppm) for mass tolerance (see `grid.ppm.from`, `grid.ppm.to` and `grid.ppm.by` parameters). If HDMSE data are used the search space is extended from ion mobility difference 0.6 to 1.6 by 0.2 (see `grid.imdiffs.from`, `grid.imdiffs.to` and `grid.imdiffs.by`). The data can be subset using using an absolute number of features (see `grid.n`) or a fixed percentage (see `grid.subset`). The pair of optimal `nsd` and `ppm` is chosen (see `grid.param.sel` parameter).

7. Fragment Matching is used to filter false-positive matches from the grid search using a default of 1 common peak for unique matches and at least a difference of 1 common peaks to choose the correct match out of non-unique matches (see `fm.minCommon` and `fm.minDelta`).

8. The intensity is corrected by a Local Polynomial Regression Fitting (`loess` function for the `stats` package) using a default `span.int` value of 0.05.

9. The quantitation EMRTs are matched using the optimised parameters.

If a master identification file is used (`master` is set to `TRUE`, default is `FALSE`), the relevant actions that have already been executed when the file was created with `makeMaster` are not repeated here.

### Value

Invisibly returns an object of class `Synapter`. Used for its side effect of creating an html report of the run in `outputdir`.

### Author(s)

Laurent Gatto, Sebastian Gibb

### References

Bond N. J., Shliaha P.V., Lilley K.S. and Gatto L. (2013) J. Prot. Research.

### Examples

    ## Not run:
    library(synapterdata)
    data(synobj2)
    output <- tempdir() ## a temporary directory
    synergise2(object = synobj2, outputdir = output, outputfile = "synapter.html")
    htmlReport <- paste0("file:///", file.path(output, "synapter.html")) ## the result report
    browseURL(htmlReport) ## open the report with default browser

    ## End(Not run)

[Package synapter version 2.14.0 Index]