Metabolomics Software Tools

Xiuxia Du, Paul Benton, Stephen Barnes

Outline

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• Introduction !!

• Software Tools for LC-MS metabolomics !

!• Software Tools for GC-MS metabolomics !

!• Software Tools for Statistical Analysis !!!!!!!

• LC-MS data analysis workflow !!!!!!!!

!• GC-MS data analysis workflow !!!!

Introduction

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data pre- processing

statistical analysis

compound identification

pathway analysis

feature extraction

statistical analysis

compound identification

pathway analysis

deconvolution

Software Tools for LC-MS

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• Commercial

• MassHunter — Agilent • MetQuest — Thermo Scientific • MetWorks — Thermo Scientific • Multiple Mass Defect Filter — Thermo Scientific • Progenesis QI — Waters • XCMSplus — Sciex

!!• and more ……

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Software Tools for LC-MS

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• Free • Insilicos Viewer !

• ProteoWizard

• SeeMS: interactive viewer for mass spec data files (Windows only)

• MSConvert: convert between various file formats

!• MZmine 2: LC-MS data processing

!• MetaboSearch: perform mass-based metabolite search simultaneously

against four major metabolite databases !

• MetaboAnalyst: a web server for metabolomics data analysis

!• and more ……

• Proprietary data formats: need vendor library functions to access !!!!!!!!!• Open data formats: free library functions to access

• netCDF • mzData • mzXML • mzML

Data format

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Company File extension

Agilent .D

Sciex .WIFF

Theomo .RAW

Waters .RAW

……

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InsilicosViewer

Insilicos Viewer

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Raw data viewer

total ion chromatogram

MS and MS/MS

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ProteoWizard

ProteoWizard: SeeMS

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• A data viewer

!!!!• Can read these open data formats

• mzML • mzXML !!

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ProteoWizard: SeeMS

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Raw data viewer

total ion chromatogram

spectrum

list of scans

ProteoWizard: MSConvert

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Data format converter

ProteoWizard: MSConvert

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• Supported data formats

• Read: open formats, vendor formats • Write: open formats

!• Filters and transformation

• msLevel • Peak picking • Zero samples • ETD filter • Threshold peak filter • Charge state predictor • Activation • Subset !!

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MZmine 2

MZmine 2

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• LC-MS metabolomics data processing, analysis, and visualization !!!!!!• Supported open data formats

• NetCDF • mzData • mzML • mzXML

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Raw data import

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list of scans in raw files • MS scans in blue • MS/MS scans in red • # sequential number • @ retention time • MS level • type of spectrum

• p = profile • c = centroid • t = thresholded

• polarity of ionization • + = positive • - = negative • ? = unknown

Raw data visualization

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Right click on the file name

brings up

Raw data visualization

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total ion chromatogram

spectrum

double click TIC

brings up

Raw data visualization

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2D view

Raw data visualization

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3D view

rotate to find the best perspective

Data pocessing

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• Workflow

• Raw data import • (optional) Raw data methods / Filtering • Peak detection • Isotopic peak grouping • (optional) Identification of fragments, adducts, and peak complexes • (optional) Normalization of retention time • Alignment • (optional) Gap filling • (optional) Normalization of peak heights / areas • (optional) Identification using database search, formula prediction, etc. • Data analysis and export !!

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Peak detection

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• Mass detection

• Centroid • Exact mass • Local maxima • Recursive threshold • Wavelet transform

!• Chromatogram building

!• Peak deconvolution

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Peak detection

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Peak detection procedure

Mass detection

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Mass detection options

click

her

e to

brin

g up

the p

aram

eter

win

dow

Mass detection

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Set mass detection parameter

Mass detection

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Set mass detection parameter

Chromatogram building

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Chromatogram builder

Chromatogram building

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Chromatogram builder — set parameters

Chromatogram building

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peak list • # ID • m/z value • @ retention time

After chromatograms are built

Chromatogram building

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To display extracted ion chromatograms

Right click on the file name

brings up

Chromatogram building

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Show chromatogram information

Chromatogram building

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Show chromatogram information

a particular extracted ion chromatogram (EIC)

Peak deconvolution

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Peak deconvolution

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Algorithms

Peak deconvolution

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Set parameters

click hereto bring up the parameter window

Peak deconvolution

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deconvolution finish

ed

Peak deconvolution

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Results

Alignment

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Retention time normalization

Alignment

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Retention time normalization: set parameters

Alignment

Join aligner

Alignment

Join aligner: set parameters

Alignment

Get aligned peak list

Alignment

Information on aligned peak list

Green: present Red: absent

Peak identification

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Options

Peak identification

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By searching custom databases

an example custom database

Peak identification

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By adduct search: set parameters

Peak identification

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Adduct search results

Peak identification

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By online database search

Peak identification

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Online database search results

Gap filling

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Options and parameters

Gap filling

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Results and visualization options

Gap filling

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Set visualization parameters

Gap filling

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Visualization

Data analysis

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Options

Data export

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Options

Data export

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Parameters

Data export

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csv format

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MetaboSearch

MetaboSearch

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Input data format

http://omics.georgetown.edu/metabosearch.html#ug

MetaboSearch

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GUI

MetaboSearch

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Steps

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Software tools for GC-MS Metabolomics

Software Tools for GC-MS

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• Commercial

• ChromaTOF® — LECO • MassHunter — Agilent

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• Free

• AMDIS: Automated Mass Spectral Deconvolution and Identification System

• NIST MS Search

• Tagfinder !!

AMDIS

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Raw data visualization

AMDIS

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Result visualization

NIST MS Search

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GUI

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Software tools for Statistical Analysis

Stats and Machine Learning

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• Commercial

• SIMCA • Mass Profiler Professional (MPP) — Agilent !

• and more …… !!!!!• Free

• MetaboAnalyst

• R !!!

MetaboAnalyst

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Other software tools

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• COMSPARI

• COMparison of SPectral And Retention Information !

• MathDAMP

• Mathematica package for Differential Analysis of Metabolite Profiles !• MSFACTs

• Metabolomics Spectral Formatting, Alignment and Conversion Tools !!and many more …… !!!!!

Thank You!