NMR Automation involves using programs like ChenoMX, Bayesil and NMRlib to automate the processing, profiling and identification of compounds from NMR spectra. ChenoMX is used to preprocess raw NMR data, profile identified compounds and build compound libraries. Bayesil and NMRlib then use these libraries to automatically process and identify compounds in spectra with minimal human input. This automation saves significant time over manual processing while improving consistency and reducing errors.

1. NMR Automation: Bayesil,
NMRlib and beyond
Philip Liu
April 11, 2013

2. NMR Automation Overview
• 1. NMR
a) history
b) Automation
• 2. ChenoMX
a) Processor
b) Profiler
c) Compound Builder
• 3. XML
• 4. NMRlib

3. 1. NMR History
• 1945 - First
experimentally
observed by Felix
Bloch (Stanford) and
Edward Purcell
(Harvard)
• 1946 - Spectra first
published in January
edition of Physical
Review
• 1952 - Nobel prize
for physics shared
by Bloch and Purcell
Felix Bloch

4. NMR (Nuclear Magnetic
Resonance)
• Nuclei of atomic isotopes (1H, 13C, 31P, 15N,
29Si etc.) interact with a static magnetic field
• Magnetic field makes spin states of nuclei
differ in energy, and transitions between
energy states can be observed
• Chemically distinct nuclei differ in resonance
frequency in the same magnetic field –

5. NMR
• Nuclei are aligned by a magnetic field
• An oscillating, perpendicular magnetic field is
applied

6. NMR
• Precession is a change in the
orientation of the rotational
axis of a rotating body.
• Dependent on rate of spin,
size and shape of gyro,
strength of gravity.
• Find out the magnetic field
strength to get the precession
frequency (frequency it takes
to get the nucleus precessing)
• 2nd magnetic field varies in
time at the same rate as the
precession - i.e. resonant with
the nuclear motion
(continuous wave NMR)
• Nuclei which are not aligned

7. • This non-equilibrium magnetisation can be induced,
generally by applying a pulse of resonant radio-
frequency close to the Larmor frequency of the nuclear
spins.
• Fourier Transform NMR, free induction decay (FID) is the
observable NMR signal generated by non-equilibrium
nuclear spin magnetisation precessing about the
magnetic field.
• The resultant oscillating magnetization vector of the
nuclei induces a current in a pickup coil, creating an
electrical signal oscillating at the NMR frequency (aka
FID).
• The FID contains the vector sum of the NMR responses
from all the excited spins
• To obtain the frequency-domain NMR spectrum (NMR
absorption intensity vs. NMR frequency) this time-
NMR - FID

8. NMR Spectra
• The chemical shift of a nucleus is the
difference between the resonance frequency of
the nucleus and a standard, relative to the
standard. (We use DSS: 4,4-dimethyl-4-
silapentane-1-sulfonic acid)

9. NMR Automation
• Why Automation?
-time: countless hours
spent processing/
profiling spectra
-inconsistencies between
different users (user
bias)
-misidentification in
profiling
-user fatigue results in
profiling errors

10. NMR Automation
Automation
-time: Faster (minutes vs.
hours)
-consistent profiling
-misidentification also
consistent, can correct
and re-run spectra
(saves time – hopefully!)
-fatigue is negligible

11. NMR Automation Workflow
ChenoMX:
Processor
NMRlib
ChenoMX:
Profiler
NMR
XML
Automation
Standards (HMDB)
Samples
BAYESIL
(Automation
Program)
.fids (raw
ChenoMX:
Compound
Builder
.fids (raw
Astoundin
g results
Library
creation
Sample
processing

12. 2. ChenoMX

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. ChenoMX Processor
• Pre-processing of raw NMR files (.fid)
• .cnx files

15. Open Processor

16. 1. Open Spectra
(.fid) - Ethanol

17. Phasing
• Phasing corrects shifts that may have occurred
in data acquisition
• Asymmetric peaks/clusters may be inverted
• Larger phase shifts may have an oscillation to
the baseline
• 3 phase angle controls: normal (1°), fine (0.1°)
and very fine (0.01°)
Sorry,
William
Shatner, not
this kind of

18. 2.
Phase

19. First order
phase
Zero order phase

20. Auto – Phase
(sometimes
works)

22. Make peaks
symmetric by
adjusting the
sliders below
(manual phasing),
accept

23. 3. Calibrate CSI
(Chemical Shift/
Shape Indicator)

24. Calibrate
automatically,
check DSS
concentration,
accept

25. 4. Region Deletion
(Water removal in
versions older than
7.0)

26. Example: Glucose
Check for peaks
you may be
deleting!!

27. You can click and
drag the blue lines/
region or type in
values.
No peaks inside
blue area, accept!

28. 5. Baseline
Correction
Removes distortion in
spectrum
Allows for accurate
quantification

29. Baseline Correction
– auto linear

30. Baseline Correction
– auto spline

31. • Move blue
dots to centre
of noise,

32. If you see wild
distortions, do not
panic. Move points to
centre of noise and it
will smooth out – hold
control and click on
the line to add points,
hold shift and click to

33. Line Broadening (Optional)
• Multiplies fid by an exponential
function before fid is Fourier
transformed
• Increases linewidth in spectrum and
smoothes out instrument noise
• Broader peaks, less noisy
• Without broadening – sharper peaks
but baseline may be noisy

34. OPTIONAL
6. Line

35. Line
broadening:
adjust Hz in

36. 7. Shim
Correction
Creates an ideal
spectrum,
removes
lineshape
distortions based

37. Shim
correction -

38. 8. Calibrate
CSI

39. Calibrate CSI
(Calibrate
Automatically)

40. Congratulations,
Processing completed,
send to Profiler!

41. ChenoMX Profiler
• Compound identification
• .cnx file

42. Type compound
name here (start
with DSS), make
sure correct
library is selected

43. Black: Spectrum
Red: Sum of profiled
compounds
Green: Subtraction line
Blue: Current compound
selected
Shortcuts: Space – automatic
fit
(bottom left will say it can’t fit
if
autofit doesn’t work)

44. These numbers will go
green if there’s a good
fit (NB: will not go
green all the time even
if fit well)
Click these numbers to
view/move different
clusters
NB: Name here is the
compound selected

45. Bottom blue arrow will
shift cluster selected on
x axis
Right side arrow will
shift all clusters on y
axis
Clicking and dragging
on the blue peak will
move all clusters x +
y

46. Fit clusters, note
concentration of
metabolite/DSS

47. Compound Builder
• Creates library files for ChenoMX
• .xcpd files

48. Open Compound
Builder

49. Overlay .cnx file
(ChenoMX file created
by processor/profiler)

50. Open .cnx

51. Edit
Compound
Details: name,
author,
magnet
frequency and
concentration

52. Ethano
l
DSS

53. Right click
peak, add new
peak to new
cluster

54. Right click,
add new peak
to existing
cluster

55. NB: Blue
denotes peaks
currently
selected

56. Automatic fit,
you can press
this multiple
times.

57. Message that
program
cannot fit any
longer, ok.

58. Adjusting peaks
manually, -
matching blue
peaks/red line
(sum line) to
match the black
lines (actual
spectra)

59. Can select and
group clusters
(Control + G)
or right click

60. Success! Save.

61. 3. XML
• Extensible Markup
Language (.xml files)
• A (document) markup
language is a modern
system for annotating
a document in a way
that is syntactically
distinguishable from
the text. [Wikipedia]
• Advantage: Machine
and Human readable

62. Conversion
from .xcpd
 XML file

64. 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>*

65. More XML
• <clusters>
• <cluster>
• <center>1.17166</center>
• <lowerBound>1.17166</
lowerBound>
• <upperBound>1.17166</
upperBound>
• <peaks>
• <peak>
• <center>1.15747</center>
Bounds limit
how far the
cluster centres
can shift
Peaks are
described by
their centres,

66. BAYESIL
• “Siamak’s Program”
• Processes NMR spectra similarly to
ChenoMX – phases, baseline corrects
etc.
• Global fit – uses area under the curve
to fit spectra
• Future improvements: UI (User
interface), flagging false positives/
negatives, concentration corrections,

67. BAYESIL

68. Automated
Human

69. 4. NMRlib
• “Roman’s Program”
• GUI (Graphical User Interface), web
based application
(nmrlib.wishartlab.com) that uses
BAYESIL to process spectra and create
XML files
• Faster than ChenoMX approach,
eliminates typographical errors
• Doesn’t allow for overlapping peaks
NMRlib

71. NMRlib
Search, sort
files by
user, date,
name,
magnet
frequency,

72. NMRlib
• Fill in fields, Name,
HMDBID, NMR Freq.
Metabolite
concentration (as
measured by
ChenoMX or
experimental), and
ratio of [metabolite]/
[DSS]
• -Spectrum files must
be .zip files and the
fid must be in a
folder named
HMDBxxxxx.fid
 eg.

73. NMRlib
Pick peaks

74. Min. peak
height:
deletes
peaks
below __
Noise factor:
higher # =
shows less
points
lower # =
shows more
Peaks picked
by program,
DSS is

75. • Peaklist: delete peaks

76. Adding peaks (click spectra)
1
2

77. Peak added!

78. Name clusters, save

79. Alternative method of
creating clusters: input range (ppm)

80. Confirm clusters

81. Run fitting!

82. Success?
Not so fast!
 Examin
e fit

83. 1. Check fit
2. Approval3. back

84. Right click
HMDB0008.x
ml, save link
as

85. Save,
SUCCESS!

86. Future
• Finish making xmls for all these
compounds
• Profile Serum (~40 cmpds) and Urine
(200+ compounds)

87. Thank you!
(You can wake up now)
• Dr. Wishart
• Igor, Trent, Rupa, Edison
• Siamak, Roman
• Wishartlab

88. Questions?

89. 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=14
6&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