NMR Automation


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NMR Automation

  1. 1. NMR Automation: Bayesil,NMRlib and beyondPhilip LiuApril 11, 2013
  2. 2. NMR Automation Overview• 1. NMR a) history b) Automation• 2. ChenoMXa) Processorb) Profilerc) Compound Builder• 3. XML• 4. NMRlib
  3. 3. 1. NMR History• 1945 - Firstexperimentallyobserved by FelixBloch (Stanford) andEdward Purcell(Harvard)• 1946 - Spectra firstpublished in Januaryedition of PhysicalReview• 1952 - Nobel prizefor physics sharedby Bloch and PurcellFelix Bloch
  4. 4. NMR (Nuclear MagneticResonance)• Nuclei of atomic isotopes (1H, 13C, 31P, 15N,29Si etc.) interact with a static magnetic field• Magnetic field makes spin states of nucleidiffer in energy, and transitions betweenenergy states can be observed• Chemically distinct nuclei differ in resonancefrequency in the same magnetic field –
  5. 5. NMR• Nuclei are aligned by a magnetic field• An oscillating, perpendicular magnetic field isapplied
  6. 6. NMR• Precession is a change in theorientation of the rotationalaxis of a rotating body.• Dependent on rate of spin,size and shape of gyro,strength of gravity.• Find out the magnetic fieldstrength to get the precessionfrequency (frequency it takesto get the nucleus precessing)• 2nd magnetic field varies intime at the same rate as theprecession - i.e. resonant withthe nuclear motion(continuous wave NMR)• Nuclei which are not aligned
  7. 7. • This non-equilibrium magnetisation can be induced,generally by applying a pulse of resonant radio-frequency close to the Larmor frequency of the nuclearspins.• Fourier Transform NMR, free induction decay (FID) is theobservable NMR signal generated by non-equilibriumnuclear spin magnetisation precessing about themagnetic field.• The resultant oscillating magnetization vector of thenuclei induces a current in a pickup coil, creating anelectrical signal oscillating at the NMR frequency (akaFID).• The FID contains the vector sum of the NMR responsesfrom all the excited spins• To obtain the frequency-domain NMR spectrum (NMRabsorption intensity vs. NMR frequency) this time-NMR - FID
  8. 8. NMR Spectra• The chemical shift of a nucleus is thedifference between the resonance frequency ofthe nucleus and a standard, relative to thestandard. (We use DSS: 4,4-dimethyl-4-silapentane-1-sulfonic acid)
  9. 9. NMR Automation• Why Automation?-time: countless hoursspent processing/profiling spectra-inconsistencies betweendifferent users (userbias)-misidentification inprofiling-user fatigue results inprofiling errors
  10. 10. NMR AutomationAutomation-time: Faster (minutes vs.hours)-consistent profiling-misidentification alsoconsistent, can correctand re-run spectra(saves time – hopefully!)-fatigue is negligible
  11. 11. NMR Automation WorkflowChenoMX:ProcessorNMRlibChenoMX:ProfilerNMRXMLAutomationStandards (HMDB)SamplesBAYESIL(AutomationProgram).fids (rawChenoMX:CompoundBuilder.fids (rawAstounding resultsLibrarycreationSampleprocessing
  12. 12. 2. ChenoMX
  13. 13. ChenoMX Processor Overview(Pre-processing)• 1. Open Processor, Open .fid file• 2. Phase• 3. Calibrate CSI• 4. Region Deletion (water removal)• 5. Baseline Correction– 6. Line Broadening (optional)• 7. Shim Correction (optional)• 8. Calibrate CSI•  Send to ChenoMX Profiler
  14. 14. ChenoMX Processor• Pre-processing of raw NMR files (.fid)• .cnx files
  15. 15. Open Processor
  16. 16. 1. Open Spectra(.fid) - Ethanol
  17. 17. Phasing• Phasing corrects shifts that may have occurredin data acquisition• Asymmetric peaks/clusters may be inverted• Larger phase shifts may have an oscillation tothe baseline• 3 phase angle controls: normal (1°), fine (0.1°)and very fine (0.01°)Sorry,WilliamShatner, notthis kind of
  18. 18. 2.Phase
  19. 19. First orderphaseZero order phase
  20. 20. Auto – Phase(sometimesworks)
  21. 21. Make peakssymmetric byadjusting thesliders below(manual phasing),accept
  22. 22. 3. Calibrate CSI(Chemical Shift/Shape Indicator)
  23. 23. Calibrateautomatically,check DSSconcentration,accept
  24. 24. 4. Region Deletion(Water removal inversions older than7.0)
  25. 25. Example: GlucoseCheck for peaksyou may bedeleting!!
  26. 26. You can click anddrag the blue lines/region or type invalues.No peaks insideblue area, accept!
  27. 27. 5. BaselineCorrectionRemoves distortion inspectrumAllows for accuratequantification
  28. 28. Baseline Correction– auto linear
  29. 29. Baseline Correction– auto spline
  30. 30. • Move bluedots to centreof noise,
  31. 31. If you see wilddistortions, do notpanic. Move points tocentre of noise and itwill smooth out – holdcontrol and click onthe line to add points,hold shift and click to
  32. 32. Line Broadening (Optional)• Multiplies fid by an exponentialfunction before fid is Fouriertransformed• Increases linewidth in spectrum andsmoothes out instrument noise• Broader peaks, less noisy• Without broadening – sharper peaksbut baseline may be noisy
  33. 33. OPTIONAL6. Line
  34. 34. Linebroadening:adjust Hz in
  35. 35. 7. ShimCorrection Creates an idealspectrum,removeslineshapedistortions based
  36. 36. Shimcorrection -
  37. 37. 8. CalibrateCSI
  38. 38. Calibrate CSI(CalibrateAutomatically)
  39. 39. Congratulations,Processing completed,send to Profiler!
  40. 40. ChenoMX Profiler• Compound identification• .cnx file
  41. 41. Type compoundname here (startwith DSS), makesure correctlibrary is selected
  42. 42. Black: SpectrumRed: Sum of profiledcompoundsGreen: Subtraction lineBlue: Current compoundselectedShortcuts: Space – automaticfit(bottom left will say it can’t fitifautofit doesn’t work)
  43. 43. These numbers will gogreen if there’s a goodfit (NB: will not gogreen all the time evenif fit well)Click these numbers toview/move differentclustersNB: Name here is thecompound selected
  44. 44. Bottom blue arrow willshift cluster selected onx axisRight side arrow willshift all clusters on yaxisClicking and draggingon the blue peak willmove all clusters x +y
  45. 45. Fit clusters, noteconcentration ofmetabolite/DSS
  46. 46. Compound Builder• Creates library files for ChenoMX• .xcpd files
  47. 47. Open CompoundBuilder
  48. 48. Overlay .cnx file(ChenoMX file createdby processor/profiler)
  49. 49. Open .cnx
  50. 50. EditCompoundDetails: name,author,magnetfrequency andconcentration
  51. 51. EthanolDSS
  52. 52. Right clickpeak, add newpeak to newcluster
  53. 53. Right click,add new peakto existingcluster
  54. 54. NB: Bluedenotes peakscurrentlyselected
  55. 55. Automatic fit,you can pressthis multipletimes.
  56. 56. Message thatprogramcannot fit anylonger, ok.
  57. 57. Adjusting peaksmanually, -matching bluepeaks/red line(sum line) tomatch the blacklines (actualspectra)
  58. 58. Can select andgroup clusters(Control + G)or right click
  59. 59. Success! Save.
  60. 60. 3. XML• Extensible MarkupLanguage (.xml files)• A (document) markuplanguage is a modernsystem for annotatinga document in a waythat is syntacticallydistinguishable fromthe text. [Wikipedia]• Advantage: Machineand Human readable
  61. 61. Conversionfrom .xcpd XML file
  62. 62. XML• File Header (red – edit these fields)• <name>Ethanol</name>• <ID>HMDB00108</ID>• <resonatingElement>hydrogen</resonatingElement>• <libraryVersion>experimental</libraryVersion>• <metabolicGroup>all</metabolicGroup>• <type>hnmrv</type>• <magnetFreq>500</magnetFreq>• <quantification>1.2863</quantification>*
  63. 63. More XML• <clusters>• <cluster>• <center>1.17166</center>• <lowerBound>1.17166</lowerBound>• <upperBound>1.17166</upperBound>• <peaks>• <peak>• <center>1.15747</center>Bounds limithow far thecluster centrescan shift Peaks aredescribed bytheir centres,
  64. 64. BAYESIL• “Siamak’s Program”• Processes NMR spectra similarly toChenoMX – phases, baseline correctsetc.• Global fit – uses area under the curveto fit spectra• Future improvements: UI (Userinterface), flagging false positives/negatives, concentration corrections,
  65. 65. BAYESIL
  66. 66. AutomatedHuman
  67. 67. 4. NMRlib• “Roman’s Program”• GUI (Graphical User Interface), webbased application(nmrlib.wishartlab.com) that usesBAYESIL to process spectra and createXML files• Faster than ChenoMX approach,eliminates typographical errors• Doesn’t allow for overlapping peaksNMRlib
  68. 68. NMRlibSearch, sortfiles byuser, date,name,magnetfrequency,
  69. 69. NMRlib• Fill in fields, Name,HMDBID, NMR Freq.Metaboliteconcentration (asmeasured byChenoMX orexperimental), andratio of [metabolite]/[DSS]• -Spectrum files mustbe .zip files and thefid must be in afolder namedHMDBxxxxx.fid eg.
  70. 70. NMRlibPick peaks
  71. 71. Min. peakheight:deletespeaksbelow __Noise factor:higher # =shows lesspointslower # =shows morePeaks pickedby program,DSS is
  72. 72. • Peaklist: delete peaks
  73. 73. Adding peaks (click spectra)12
  74. 74. Peak added!
  75. 75. Name clusters, save
  76. 76. Alternative method ofcreating clusters: input range (ppm)
  77. 77. Confirm clusters
  78. 78. Run fitting!
  79. 79. Success?Not so fast! Examine fit
  80. 80. 1. Check fit2. Approval3. back
  81. 81. Right clickHMDB0008.xml, save linkas
  82. 82. Save,SUCCESS!
  83. 83. Future• Finish making xmls for all thesecompounds• Profile Serum (~40 cmpds) and Urine(200+ compounds)
  84. 84. Thank you!(You can wake up now)• Dr. Wishart• Igor, Trent, Rupa, Edison• Siamak, Roman• Wishartlab
  85. 85. Questions?
  86. 86. References• http://chemnmr.colorado.edu/moreinfo/whatisnmr.html• http://www.google.ca/imgres?imgurl=http%3A%2F%2Fwww.riken.jp%2Fengn%2Fr-world%2Finfo%2Frelease%2Fnews%2F2003%2Faug%2Fimage%2Fefg-2.jpg&imgrefurl=http%3A%2F%2Fwww.riken.jp%2Fengn%2Fr-world%2Finfo%2Frelease%2Fnews%2F2003%2Faug%2Findex.html&docid=IDBu8C4qtKDVSM&tbnid=npBbvuV7tHmVLM&w=246&h=146&ei=mCZnUc2DB6ayigKl04GwDQ&ved=0CAUQxiAwAw&iact=ricl• http://edwebproject.org/india/pics/swing.jpg• http://www.abc.net.au/science/articles/2011/03/29/3175747.htm• http://www.planetseed.com/node/15291• http://en.wikipedia.org/wiki/File:Earth_precession.svg• http://www.physiopro.co.za/the-head-work-behind-your-headache/#• http://4.bp.blogspot.com/-roZe_7I5lJ4/TWxGXMuRoDI/AAAAAAAABSE/fe5qFP_iGBw/s1600/BERTI_robot.jpg• http://i.current.com/images/asset/900/821/06/4Fo01x.jpg• http://i.telegraph.co.uk/multimedia/archive/00802/460-basil_802346c.jpg• http://www.chenomx.com/support/img/page58/Chenomx%20NMR%20Suite%207.1.pdf