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Metabolomics Software Tools Xiuxia Du, Paul Benton, Stephen Barnes Outline ! • Introduction ! ! ! ! • Software Tools for LC-MS metabolomics ! ! ! ! • !Software ! ! Tools for GC-MS metabolomics ! • !Software Tools for Statistical Analysis !! !! !! !! ! !! !! 2 Introduction • !LC-MS data analysis workflow ! ! ! ! ! ! ! data pre! ! processing ! ! statistical analysis compound identification pathway analysis ! ! ! ! ! • !GC-MS data analysis workflow ! !! feature ! extraction ! ! deconvolution statistical analysis compound identification pathway analysis 3 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 ! ! ! ! ! • and more …… ! ! ! ! !! !! ! ! ! 4 Software Tools for LC-MS • !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 …… 5 Data format • Proprietary data formats: need vendor library functions to access ! Company File extension ! Agilent .D ! Sciex .WIFF ! Theomo .RAW ! Waters .RAW ! ! …… ! ! • Open data formats: free library functions to access • • • • netCDF mzData mzXML mzML 6 InsilicosViewer 7 Insilicos Viewer Raw data viewer ! ! ! total ion chromatogram ! ! ! ! ! ! ! MS and MS/MS ! ! ! ! ! 8 ProteoWizard 9 ProteoWizard: SeeMS • A data viewer ! ! ! ! • Can read these open data formats • mzML • mzXML ! ! ! ! ! ! ! 10 ProteoWizard: SeeMS Raw data viewer ! ! ! ! ! total ion chromatogram ! ! ! ! ! spectrum ! ! ! ! list of scans ! 11 ProteoWizard: MSConvert Data format converter 12 ProteoWizard: MSConvert • 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 ! ! ! 13 MZmine 2 14 MZmine 2 ! metabolomics data processing, analysis, and visualization • LC-MS ! ! ! ! ! ! ! ! ! ! ! ! • !Supported open data formats ! • NetCDF ! • mzData ! • mzML ! • mzXML ! ! ! ! ! 15 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 16 Raw data visualization brings up Right click on the file name 17 Raw data visualization double click TIC total ion chromatogram brings up spectrum 18 Raw data visualization ! ! ! ! 2D view ! ! ! ! ! ! ! ! ! ! ! 19 Raw data visualization rotate to find the best perspective 3D view 20 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 ! ! ! ! 21 Peak detection • Mass detection • • • • • Centroid Exact mass Local maxima Recursive threshold Wavelet transform ! • Chromatogram building ! • Peak deconvolution ! ! ! ! ! ! 22 Peak detection Peak detection procedure ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 23 Mass detection Mass detection options ! ! ! ! ow ! in d ! er w ! ar am et ! ep ! up th ! br in g ! ! re t he k cl ic ! ! o ! 24 Mass detection Set ! mass detection parameter ! ! ! ! ! ! ! ! ! ! ! ! ! ! 25 Mass detection Set ! mass detection parameter ! ! ! ! ! ! ! ! ! ! ! ! ! ! 26 Chromatogram building Chromatogram builder ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 27 Chromatogram building Chromatogram builder — set parameters ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 28 Chromatogram building After chromatograms are built ! ! ! ! ! ! ! ! ! peak list • # ID • m/z value • @ retention time ! ! ! ! ! ! 29 Chromatogram building To display extracted ion chromatograms brings up Right click on the file name 30 Chromatogram building Show chromatogram information ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 31 Chromatogram building Show chromatogram information ! ! ! ! ! ! ! a particular extracted ion chromatogram (EIC) ! ! ! ! ! ! ! ! 32 Peak deconvolution 33 Peak deconvolution Algorithms ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 34 Peak deconvolution Set parameters click here to bring up the parameter window 35 Peak deconvolution ! ! ! ! ! ! ! ! de i f n o i t u l o v n co d e h nis ! ! ! ! ! ! ! 36 Peak deconvolution Results ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 37 Alignment Retention time normalization ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 38 Alignment 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 Options 44 Peak identification By searching custom databases an example custom database 45 Peak identification By adduct search: set parameters 46 Peak identification Adduct search results 47 Peak identification By online database search 48 Peak identification Online database search results 49 Gap filling Options and parameters 50 Gap filling Results and visualization options 51 Gap filling Set visualization parameters 52 Gap filling Visualization 53 Data analysis Options 54 Data export Options 55 Data export Parameters 56 Data export csv format 57 MetaboSearch 58 MetaboSearch Input data format 59 http://omics.georgetown.edu/metabosearch.html#ug MetaboSearch GUI 60 MetaboSearch Steps 61 Software tools for GC-MS Metabolomics 62 Software Tools for GC-MS • !Commercial • !ChromaTOF® — LECO ! • MassHunter — Agilent ! ! ! ! ! ! ! ! ! ! ! • !Free ! • AMDIS: Automated Mass Spectral Deconvolution and Identification System NIST MS Search !• ! • Tagfinder ! ! ! ! 63 AMDIS Raw data visualization ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 64 AMDIS Result visualization ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 65 NIST MS Search GUI ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 66 Software tools for Statistical Analysis 67 Stats and Machine Learning • !Commercial • !SIMCA ! • Mass Profiler Professional (MPP) — Agilent ! ! ! • and more …… ! ! ! ! ! ! ! ! ! ! • !Free ! • MetaboAnalyst !• R ! ! ! ! ! 68 MetaboAnalyst 69 Other software tools • !COMSPARI • !COMparison of SPectral And Retention Information ! ! • !MathDAMP ! • Mathematica package for Differential Analysis of Metabolite Profiles ! ! • !MSFACTs ! • Metabolomics Spectral Formatting, Alignment and Conversion Tools ! ! ! ! ! many more …… and ! !! ! ! ! 70 Thank You!