The SIRIUS commandline tool can be called via the “binary/startscript” by simply running the command in the commandline:

sirius --help

NOTE: It is usually a good idea to use the --help option to obtain an overview of the available commands and options. This will guarantee that you will have a description of the commands that directly matches your SIRIUS version.

Introduction

The SIRIUS commandline program is designed as a toolbox that provides different tools (subcommands) for metabolite identification. These tools can be concatenated to toolchains to compute multiple analysis steps at once. We distinguish the following types of subcommands:

Subtool types

  • CONFIGURATION: The config tool can be executed before every toolchain or standalone tool to set all configurations available in SIRIUS from the command line.

  • STANDALONE: Tools that run Standalone and cannot be concatenated with other subtools. These are usually tools for configuration or data management purposes. E.g. modifying project-space or exporting MGF files.

  • PREPROCESSING: Tools that prepare input data to be compatible with SIRIUS. E.g. lcms-align for feature detection and alignment.

  • COMPOUND TOOL: Tools that analyze each compound (instance) of the dataset individually and can be concatenated with other tools. E.g. formula annotation, structure database search or canopus compound class prediction.

  • DATASET TOOL: Tools that analyze all compounds (instances) of the dataset simultaneously and can be concatenated with other tools. E.g. dataset-wide molecular formula annotatio with zodiac.

Each subtool can also be called with the --help option to get a documentation about the available options and possible follow-up commands in a toolchain. For the formula tool the command would be:

sirius formula --help

Basic principles

The SIRIUS CLI toolbox can also be considered as a rudimentary workflow engine (toolchains) that works according the following principles:

  • It only executes subtools that are specified in the command.
  • It skips computation of a compound if a mandatory input from a previous step (subtool) is missing.
  • Without an additional parameter it does not override existing results. Compounds where the result to be computed already exist will be skipped.
  • If --recompute is specified, existing results will be replaced with new ones for all subtools that are specified in the command.
  • If a subtool is recomputed (--recompute), the results of subtools that depend on the results of the recomputed subtool will be lost too. This is necessary because they would not be consistent with the newly computed results anymore.

Exemplary scenarios

  1. Compute missing results without recompute: Assume we have computed (formula, structure) results for compounds <600Da. We can now run the workflow (formula, structure, canopus) on the same project-space without restricting the precursor mass to <600Da. Since for the <600Da compounds results of the formula and structure tools already exist, they will be skipped and not recomputed. However, canopus will be executed for all compounds.
  2. Recompute all results: If we do the same with --recompute, all results will be recomputed.
  3. Recompute only one subtool: Assume we have a project-space with complete results for formula, fingerprint, structure, canopus and want recompute the structure results because we have used the wrong parameters. We can now execute sirius -i <projekt> --recompute structure -db mydb. This will result in recomputing all structure results but without recomputing the formula results. Even the canopus results will not be lost since they only depend on the fingerprint results but not on the strucure tool results. Note: If we would do the same with the fingerprint tool, both, the structure and the canopus results would be lost.
  4. Proceed interrupted computations: Let’s say we have an interrupted computation. We can just rerun the same command to proceed the computation. SIRIUS will skip computation for existing results and compute only the missing ones.
  5. Special Case zodiac: Since the zodiac tool is not calculated on a per compound basis but on the whole dataset, it will always be recomputed completely as soon as not all compounds contain zodiac results.
  6. Visualize results in the GUI: Results of all subtools that produce a project-space as output (--output option), can also be opened in the GUI for visualization, modification or even computation.



Commands

In the following, the most important (sub)commands and options are described shortly.

LCMS-align: Feature detection and feature alignment [Preprocessing]

The lcms-align tool allows us to import multipe mzML/mzXML files into SIRIUS. It performs feature detection and feature alignment based on the MS/MS spectra and creates a SIRIUS project-space which is then used to execute followup analysis steps:

sirius -i <mzml(s)> -o <projectspace> lcms-run formula

SIRIUS: Identifying Molecular Formulas [Compound Tool]

One main purpose of SIRIUS is identifying the molecular formula of a measured ion. For this task SIRIUS provides the formula tool. The most basic way to use the formula tool is with the generic text/CSV input:

sirius [OPTIONS] -1 <MS FILE> -2 <MS/MS FILES comma separated> -z <PARENTMASS> --adduct <adduct> --output <projectspace> formula

Where MS FILE and MS/MS FILE are either CSV or MGF files. If MGF files are used, you might omit the -z option. If you omit the --adduct option, [M+?]+ is used as default. It is also possible to give a list of MS/MS files if you have several measurements of the same compound with different collision energies. SIRIUS will merge these MS/MS spectra into one spectrum.

The more common and recommended way is using input files in .ms or .mgf format (with MSLEVEL and PEPMASS meta information). Such files contain all spectra for a compound together with their meta data. They can also contain multiple compounds per file. Further SIRIUS is able to crawl an input directory for supported files:

sirius [OPTIONS] --input <inputFile> --output <projectspace> formula [OPTIONS]

SIRIUS will pick the meta information (parentmass, ionization etc.) from the .ms files in the given directory. This allows SIRIUS to run in batch mode (analyzing multiple compounds without starting a new jvm process every time).

Results such as scored molecular formula candidates and corresponding fragmentation trees in .json format are written in the <projectspace>. If the command write-summaries is used, SIRIUS will output a summary file on compound level containing among others the ‘rank’, ‘molecularFormula’, ‘adduct’, ‘precursorFormula’, ‘rankingScore’, ‘SiriusScore’, ‘TreeScore’, ‘IsotopeScore’, ‘numExplainedPeaks’, ‘explainedIntensity’, ‘medianMassErrorFragmentPeaks(ppm)’, ‘medianAbsoluteMassErrorFragmentPeaks(ppm)’, ‘massErrorPrecursor(ppm)’ and a summary containing the top hits for all compounds on project level.

The SiriusScore is the sum of the TreeScore and the IsotopeScore. The tool uses the SiriusScore for ranking. If the IsotopeScore is negative, it is set to zero. If at least one IsotopeScore is greater than 10, the isotope pattern is considered to have good quality and only the candidates with best isotope pattern scores are selected for further fragmentation pattern analysis.

Computing fragmentation trees

If you already know the correct molecular formula and just want to compute a fragmentation tree, you can specify the formula using --formulas. SIRIUS will then only compute a tree for this molecular formula. If your input data is in .ms format, the molecular formula might be already specified within the file. Note: the --formulas options can also be used to specify a comma-separated list of candidate molecular formulas.

sirius -i <input> --output <projectspace> formula --formulas <formula>

Instrument-specific parameters

Datasets have different mass errors, level of noise and accuracy of isotope pattern intensities, depending, among others, on instrument type and setup. By default, SIRIUS uses a profile for Q-TOF data with 10 ppm mass deviation. This should not be interpreted as a Q-TOF-only profile, but is often a good default profile even for data from other instruments. However, if you are certain that your data has mass errors much below 10 ppm - because if was measured on Orbitrap or FT-ICR - you should probably specify more stringent parameters. Adjustments are also necessary if the data is expected to have even higher mass errors. Both can be accomplished by specifying a different profile and mass deviations.

You may be familiar with the profile option from the GUI. Using the CLI, you can specify -p <name> to either select qtof (default) or orbitrap. orbitrap will mainly use a different mass deviation of 5 ppm and slightly different settings for isotope scoring. For FT-ICR data, we recommend to use the orbitrap profile and additionally specify a lower mass deviation, as explained in the following.

You can specify the maximum allowed mass deviations for MS1 and MS2 and separately:

sirius -i <input> --output <projectspace> formula -p orbitrap --ppm-max 2 --ppm-max-ms2 5

ZODIAC: Improve Molecular Formula Identifications [Dataset Tool]

If your input data is derived from a biological sample or any other set of derivatives, similarities between different compounds can be leveraged to improve molecular formula annotation of the individual compounds. ZODIAC builds a similarity network between molecular formula candidates of all compounds that where computed via the formula tool and re-ranks these candidates using Bayesian statistics (Gibbs Sampling). This decreases error rates (of top 1 candidates) by approximately 2 fold — on challenging datasets that contain many large compounds, improvements can be much more dramatic.

The zodiac tool can be executed after the formula tool without the need of many parameters:

sirius -i <input> -o <projectspace> formula -c 50 zodiac

When using ZODIAC, it is reasonable to increase the maximum number of formula candidates (-c) that are stored after running formula. These candidates are input to ZODIAC. If the correct candidate is missing, ZODIAC cannot recover it. In order to reduce memory consumption and running time, ZODIAC uses a dynamic number of candidates per compound based on the m/z — the idea is, for low-mass compounds the correct molecular formula is much more likely to be in the, say, top 10. By default, ZODIAC uses 10 candidates for compounds with m/z lower equal to 300 (--considered-candidates-at-300 10) and 50 candidates for compounds with m/z greater equal to 800 (--considered-candidates-at-800 50). In between these thresholds the number of candidates is calculated by interpolation.

The density of the ZODIAC network mainly depends on two parameters: --edge-threshold (default:0.95) and --minLocalConnections (default:10). The edge threshold defines the ratio of all possible edges between candidates that are discarded. Because most formula candidates are incorrect (there is only one correct candidate per compound) we assume most edges are spurious and we throw away the 95% edges with lowest score. However, to prevent compounds being disconnected completely from the rest of the network, we discard edges in such a way that one candidate per compound is connected to at least --minLocalConnections other compounds. This introduces an individual edge score threshold for each compound. However, when using --minLocalConnections, ZODIAC first has to create the complete network and filter edges afterwards. Thus, ZODIAC may consume a large amount of system memory.

For very large datasets, the ZODIAC network may not fit in 1TB system memory and more. Please, perform a feature alignment between our LC-MS/MS runs to reduce the number of compounds and thus reduce the size of the ZODIAC network. If this is still not sufficient, memory consumption can be dramatically decreased by setting --minLocalConnections=0. This will allow ZODIAC to filter low weight edges on the fly when creating the network. Use this setting with care, since it can result in a badly connected network that may decrease performance:

sirius -i <input> -o <projectspace> formula -c 50 zodiac --minLocalConnections 0 --edge-threshold 0.99

CSI:FingerID: Predicting molecular fingerprints [Compound Tool]

Molecular fingerprints can be predicted via fingerprints command after molecular formula candidates have been calculated running formula. Fingerprint prediction is part of CSI:FingerID. A fingerprint is predicted based on a specific molecular formula candidate (with corresponding fragmentation tree). By default, fingerprints are predicted for multiple good scoring formula candidates by applying a soft score-threshold on the SIRIUS score.

sirius -i <input> -o <projectspace> formula fingerprint

CSI:FingerID: Identifying Molecular Structures [Compound Tool]

Using the structure tool we can search with CSI:FingerID for molecular structures in a structure database. To run structure database search, molecular fingerprints need to be predicted in advance by running the fingerprint tool first. You might also want to run the zodiac tool for improved formula ranking if your data is derived from a biological sample or any other set of derivatives.

With --databases we can specify the database CSI:FingerID should search in. Available are, among other pubchem and bio.

When structure search was performed, the write-summaries tool will generate a structure_candidates.csv for each compound containing an ordered candidate list of structures with the CSI:FingerID score. Furthermore, a projectspace-wide compound_identification.csv file will be generated containing the top candidate structure for each compound ordered by their confidence score.

sirius -i <input> -o <projectspace> formula fingerprint structure --database pubchem

When running structure together with zodiac the command could look like this:

sirius -i <input> -o <projectspace> formula -c 50 zodiac fingerprint structure --database bio

CANOPUS: Database-free Compound Classes Prediction [Compound Tool]

The canopus tool allows us to directly predict compound classes based on the probabilistic molecular fingerprint that was predicted by CSI:FingerID (fingerprint command). Notably, canopus can even provide compound class information for unidentified compounds with no hit in a structure database:

sirius -i <input> -o <projectspace> formula fingerprint canopus

PASSATUTTO: Decoy Spectra from Fragmentation Trees [Compound Tool]

The passattuto tool allows us to compute high quality decoy spectra from fragmentation trees provided by the formula tool. Assume we are using a spectral library as input us can easily create a decoy database based on these spectra:

sirius -i <spectral-lib> -o <projectspace> formula passatutto

If no molecular formulas are annotated to the input spectra the best scoring candidate will be used for decoy computation instead.