This document summarizes an analysis comparing the primary leaf metabolites of pumpkin and tomatillo plants. The goal was to carry out statistical analyses, hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (O-PLS-DA) on metabolite profile data from pumpkin and tomatillo leaf samples. Both the HCA and PCA suggested that the treatment effect on metabolite profiles was minor compared to differences between species. A PLS-DA model was validated and found to have outstanding performance in discriminating between pumpkin and tomatillo leaf metabolites. Top discriminating metabolites between the species were then identified.

1. Biology
Chemistry
Comparison of Leaf Metabolites
Informatics
Comparison of pumpkin and
tomatillo leaf primary
metabolites
Goal:
Carry out a statistical, HCA, PCA and O-/PLS-DA
analyses comparing leaf primary metabolite profiles
(Used DATA: Pumpkin and Tomatillo 1.csv)
Cucurbita pepo
Physalis philadelphica

2. Biology
Chemistry
Comparison of Leaf Metabolites
Informatics
Comparison of pumpkin and
tomatillo leaf primary
metabolites
Steps:
1.Identify analysis strategy (hint: use HCA
and PCA)
2.Conduct statistical comparison
3.Identify top multivariate discriminants

3. Data Exploration
Biology
Chemistry
Comparison of Leaf Metabolites
Informatics
Steps:
1. Identify the effect of treatment on species differences
• Use HCA
• PCA
Exercise:
Can different treatments be analyzed together to
identify species differences?

4. HCA clustering of samples
Biology
Chemistry
Comparison of Leaf Metabolites
Informatics

5. Biology
Chemistry
Comparison of Leaf Metabolites
Informatics
raw
PCA:
comparison of
pretreatments
Mean centered
Auto scaled

6. Biology
Chemistry
Identify Analysis
Strategy
Informatics
Comparison of Leaf Metabolites
Analysis Options:
If the treatment is a minor effect compared to species differences:
•
two-sample t-Test for Species
If the treatment is a has a considerable effect compared to species
differences:
•
two-way ANOVA for Species + treatment + interaction
(species/treatment)
If the treatment has a similar effect size to species differences:
•
Eliminate one treatment type from analysis and use t-Test
Conclusions:
•
Both PCA and HCA analyses suggests the treatment effect is minor
•
Using 12 compared to 6 samples per group will increased study power

7. Comparison PLS-DA to O-PLS-DA
Biology
Chemistry
Informatics
O-PLS-DA is only useful over PLS-DA when the axis of separation
between two groups spans >1 dimension
Comparison of Leaf Metabolites
PLS-DA
O-PLS-DA

8. Biology
Chemistry
Comparison of Leaf Metabolites
Informatics
Validation of PLS-DA model for
discrimination between pumpkin
and tomatillo leaf metabolites
Outstanding model
performance, highly unlikely
by random chance

9. Biology
Chemistry
Comparison of Leaf Metabolites
Informatics
Identification of top multivariate
discriminants between pumpkin and
tomatillo leaf primary metabolites
Could also select
from increasing
and decreasing
metabolites
separately