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Metabolomics Software Tools
Xiuxia Du, Paul Benton, Stephen Barnes
Outline
2
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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
3
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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
4
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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
5
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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
6
Company File extension
Agilent .D
Sciex .WIFF
Theomo .RAW
Waters .RAW
……
7
InsilicosViewer
Insilicos Viewer
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Raw data viewer
total ion chromatogram
MS and MS/MS
9
ProteoWizard
ProteoWizard: SeeMS
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• A data viewer
!!!!• Can read these open data formats
• mzML • mzXML !!
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ProteoWizard: SeeMS
11
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Raw data viewer
total ion chromatogram
spectrum
list of scans
ProteoWizard: MSConvert
12
Data format converter
ProteoWizard: MSConvert
13
• 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 !!
!
14
MZmine 2
MZmine 2
15
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• LC-MS metabolomics data processing, analysis, and visualization !!!!!!• Supported open data formats
• NetCDF • mzData • mzML • mzXML
!!!!
Raw data import
16
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
17
Right click on the file name
brings up
Raw data visualization
18
total ion chromatogram
spectrum
double click TIC
brings up
Raw data visualization
19
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2D view
Raw data visualization
20
3D view
rotate to find the best perspective
Data pocessing
21
• 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 !!
!!
Peak detection
22
• 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
24
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Mass detection options
click
her
e to
brin
g up
the p
aram
eter
win
dow
Mass detection
25
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Set mass detection parameter
Mass detection
26
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Set mass detection parameter
Chromatogram building
27
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Chromatogram builder
Chromatogram building
28
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Chromatogram builder — set parameters
Chromatogram building
29
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peak list • # ID • m/z value • @ retention time
After chromatograms are built
Chromatogram building
30
To display extracted ion chromatograms
Right click on the file name
brings up
Chromatogram building
31
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Show chromatogram information
Chromatogram building
32
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Show chromatogram information
a particular extracted ion chromatogram (EIC)
Peak deconvolution
33
Peak deconvolution
34
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Algorithms
Peak deconvolution
35
Set parameters
click hereto bring up the parameter window
Peak deconvolution
36
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deconvolution finish
ed
Peak deconvolution
37
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Results
Alignment
38
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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
44
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
47
Adduct search results
Peak identification
48
By online database search
Peak identification
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Online database search results
Gap filling
50
Options and parameters
Gap filling
51
Results and visualization options
Gap filling
52
Set visualization parameters
Gap filling
53
Visualization
Data analysis
54
Options
Data export
55
Options
Data export
56
Parameters
Data export
57
csv format
58
MetaboSearch
MetaboSearch
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Input data format
http://omics.georgetown.edu/metabosearch.html#ug
MetaboSearch
60
GUI
MetaboSearch
61
Steps
62
Software tools for GC-MS Metabolomics
Software Tools for GC-MS
63
!!
!!!!!!!!!!!!!
• Commercial
• ChromaTOF® — LECO • MassHunter — Agilent
!!!!!!
• Free
• AMDIS: Automated Mass Spectral Deconvolution and Identification System
• NIST MS Search
• Tagfinder !!
AMDIS
64
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Raw data visualization
AMDIS
65
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Result visualization
NIST MS Search
66
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GUI
67
Software tools for Statistical Analysis
Stats and Machine Learning
68
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• Commercial
• SIMCA • Mass Profiler Professional (MPP) — Agilent !
• and more …… !!!!!• Free
• MetaboAnalyst
• R !!!
MetaboAnalyst
69
Other software tools
70
!!
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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!
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