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Metabolomics Software Tools
Xiuxia Du, Paul Benton, Stephen Barnes
Outline
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• Introduction
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• Software Tools for LC-MS metabolomics
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• !Software
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Tools for GC-MS metabolomics
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• !Software
Tools for Statistical Analysis
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Introduction
• !LC-MS data analysis workflow
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! ! data pre! ! processing
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statistical
analysis
compound
identification
pathway
analysis
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• !GC-MS data analysis workflow
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!! feature
! extraction
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deconvolution
statistical
analysis
compound
identification
pathway
analysis
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Software Tools for LC-MS
• !Commercial
• !MassHunter — Agilent
! • MetQuest — Thermo Scientific
! • MetWorks — Thermo Scientific
! • Multiple Mass Defect Filter — Thermo Scientific
! • Progenesis QI — Waters
! • XCMSplus — Sciex
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! • and more ……
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Software Tools for LC-MS
• !Free
• !Insilicos
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Viewer
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• SeeMS: interactive viewer for mass spec data files (Windows only)
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MSConvert: convert between various file formats
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• MZmine 2: LC-MS data processing
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MetaboSearch: perform mass-based metabolite search simultaneously
against four major metabolite databases
! • MetaboAnalyst: a web server for metabolomics data analysis
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and more ……
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Data format
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Proprietary data formats: need vendor library functions to access
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Company
File extension
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Agilent
.D
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Sciex
.WIFF
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Theomo
.RAW
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Waters
.RAW
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Open data formats: free library functions to access
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netCDF
mzData
mzXML
mzML
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InsilicosViewer
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Insilicos Viewer
Raw
data viewer
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total ion chromatogram
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MS and MS/MS
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ProteoWizard
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ProteoWizard: SeeMS
• A data viewer
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• Can read these open data formats
• mzML
• mzXML
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ProteoWizard: SeeMS
Raw
data viewer
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total ion chromatogram
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spectrum
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list of scans
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ProteoWizard: MSConvert
Data format converter
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ProteoWizard: MSConvert
• Supported data formats
• Read: open formats, vendor formats
• Write: open formats
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Filters and transformation
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msLevel
Peak picking
Zero samples
ETD filter
Threshold peak filter
Charge state predictor
Activation
Subset
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MZmine 2
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MZmine 2
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metabolomics data processing, analysis, and visualization
• LC-MS
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• !Supported open data formats
! • NetCDF
! • mzData
! • mzML
! • mzXML
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Raw data import
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
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Raw data visualization
brings up
Right click on the file name
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Raw data visualization
double
click TIC
total ion chromatogram
brings up
spectrum
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Raw data visualization
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2D view
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Raw data visualization
rotate to find the
best perspective
3D view
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Data pocessing
• 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
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Centroid
Exact mass
Local maxima
Recursive threshold
Wavelet transform
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Chromatogram building
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Peak deconvolution
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Peak detection
Peak
detection procedure
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Mass detection
Mass
detection options
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Mass detection
Set
! mass detection parameter
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Mass detection
Set
! mass detection parameter
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Chromatogram building
Chromatogram
builder
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Chromatogram building
Chromatogram
builder — set parameters
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Chromatogram building
After
chromatograms are built
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peak list
• # ID
• m/z value
• @ retention time
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Chromatogram building
To display extracted ion chromatograms
brings up
Right click on the file name
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Chromatogram building
Show
chromatogram information
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Chromatogram building
Show
chromatogram information
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a particular
extracted ion chromatogram (EIC)
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Peak deconvolution
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Peak deconvolution
Algorithms
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Peak deconvolution
Set parameters
click here
to bring up the parameter window
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Peak deconvolution
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de
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Peak deconvolution
Results
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Alignment
Retention
time normalization
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Alignment
Retention
time normalization: set parameters
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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
Options
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Peak identification
By searching custom databases
an example custom database
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Peak identification
By adduct search: set parameters
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Peak identification
Adduct search results
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Peak identification
By online database search
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Peak identification
Online database search results
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Gap filling
Options and parameters
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Gap filling
Results and visualization options
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Gap filling
Set visualization parameters
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Gap filling
Visualization
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Data analysis
Options
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Data export
Options
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Data export
Parameters
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Data export
csv format
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MetaboSearch
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MetaboSearch
Input data format
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http://omics.georgetown.edu/metabosearch.html#ug
MetaboSearch
GUI
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MetaboSearch
Steps
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Software tools for GC-MS Metabolomics
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Software Tools for GC-MS
• !Commercial
• !ChromaTOF® — LECO
! • MassHunter — Agilent
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• !Free
! • AMDIS: Automated Mass Spectral Deconvolution and Identification
System
NIST MS Search
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! • Tagfinder
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AMDIS
Raw
data visualization
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AMDIS
Result
visualization
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NIST MS Search
GUI
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Software tools for Statistical Analysis
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Stats and Machine Learning
• !Commercial
• !SIMCA
! • Mass Profiler Professional (MPP) — Agilent
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• !Free
! • MetaboAnalyst
!• R
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MetaboAnalyst
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Other software tools
• !COMSPARI
• !COMparison of SPectral And Retention Information
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• !MathDAMP
! • Mathematica package for Differential Analysis of Metabolite Profiles
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• !MSFACTs
! • Metabolomics Spectral Formatting, Alignment and Conversion Tools
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! many more ……
and
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70
Thank You!
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