Volcano plot
84 × 70 mm · editable SVG text
Figure standard. Lipidomics volcano: native 84 × 70 mm; metabolomics complete-name volcano: 170 mm wide with content-responsive height, unchanged P<0.05 and |log2FC/effect|≥0.585 thresholds, V19 candidate order retained within each selection stratum, highlighted points use a 1.5-mm color-core diameter with no outline, 7 pt labels without background boxes, 0.7 pt leader lines. Heatmap: ~120-mm Option-3 wide format, row Z-score, no values inside cells; Top5/Top10 are selected separately in the higher/positive and lower/negative directions, using only candidates meeting P<0.05 and |effect|≥0.585. If a direction has fewer candidates, the actual number is shown without padding. KEGG plots use the correctly matched comparison-specific vendor tables; the source tables contain hsa pathway identifiers and should be rerun with a mouse-specific background before final mechanistic claims. † denotes a putative vendor-library candidate requiring authentic-standard confirmation. All selected metabolomics features are displayed with complete, wrapped candidate names; m/z and retention time are retained only in the annotation audit table. Open annotation audit · Download annotation source data.
POS
Lipidomics · positive ion mode
POS · NF vs FR
Volcano direction: FR / NF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
n=10
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
POS · KONF vs KOFR
Volcano direction: KOFR / KONF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
POS · NF vs KONF
Volcano direction: KONF / NF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
POS · FR vs KOFR
Volcano direction: KOFR / FR; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
POS · genotype × fear interaction
Four-group interaction model; heatmaps include NF, FR, KO-No fear, and KO-Fear.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
NEG
Lipidomics · negative ion mode
NEG · NF vs FR
Volcano direction: FR / NF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
NEG · KONF vs KOFR
Volcano direction: KOFR / KONF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
NEG · NF vs KONF
Volcano direction: KONF / NF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
NEG · FR vs KOFR
Volcano direction: KOFR / FR; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
NEG · genotype × fear interaction
Four-group interaction model; heatmaps include NF, FR, KO-No fear, and KO-Fear.
Volcano plot
84 × 70 mm · editable SVG text
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Met
Metabolomics
Met · FLOX-No fear vs FLOX-Fear
Volcano direction: FR / NF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
170 mm wide · complete wrapped editable SVG text · Top10 points 1.50 mm
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
KEGG enrichment
Correct comparison-specific source table · top pathways by adjusted P
Met · KO-No fear vs KO-Fear
Volcano direction: KOFR / KONF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
170 mm wide · complete wrapped editable SVG text · Top10 points 1.50 mm
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
KEGG enrichment
Correct comparison-specific source table · top pathways by adjusted P
Met · FLOX-No fear vs KO-No fear
Volcano direction: KONF / NF; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
170 mm wide · complete wrapped editable SVG text · Top10 points 1.50 mm
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
KEGG enrichment
Correct comparison-specific source table · top pathways by adjusted P
Met · FLOX-Fear vs KO-Fear
Volcano direction: KOFR / FR; heatmaps show separately ranked strict-threshold higher and lower candidates without nonsignificant padding.
Volcano plot
170 mm wide · complete wrapped editable SVG text · Top10 points 1.50 mm
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
KEGG enrichment
Correct comparison-specific source table · top pathways by adjusted P
Met · genotype × fear interaction
Four-group interaction model; heatmaps include NF, FR, KO-No fear, and KO-Fear.
Volcano plot
170 mm wide · complete wrapped editable SVG text · Top10 points 1.50 mm
Heatmap Top5 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
Heatmap Top10 per direction
Strict threshold · higher/positive + lower/negative · all rows labeled
QC
Supplementary quality control
Unsupervised QC standard. PCA uses every filtered feature without outcome-driven or VIP-based selection. Intensities are log2 sample-median normalized, mean-centered and Pareto-scaled. Pooled QC is included in panel A; panel B recomputes PCA using biological samples only and adds descriptive 80% group data ellipses. The ellipses show within-group dispersion, are not confidence intervals or significance tests, and are omitted for pooled QC and injection-order PCA. No PCA-based sample exclusions are used. Lipidomics has three pooled-QC injections, so injection-order traces are descriptive and are not treated as evidence of a fitted drift correction.
Figures S2-S4 · dataset-specific QC
Each supplementary figure contains all-sample PCA, biological-only PCA, PCA colored by injection order, pooled-QC RSD, total signal by injection order, and a sample-correlation heatmap.
Lipidomics - positive ion mode
Six-panel supplementary QC · biological PCA includes editable 80% data ellipses
Lipidomics - negative ion mode
Six-panel supplementary QC · biological PCA includes editable 80% data ellipses
Metabolomics
Six-panel supplementary QC · biological PCA includes editable 80% data ellipses
QC summary
Values are recomputed from the local filtered peak-intensity matrices and pooled-QC samples.
| Dataset | Biological n | QC n | Raw features | Filtered features | Median QC-RSD | RSD ≤30% |
|---|---|---|---|---|---|---|
| POS | 19 | 3 | 1078 | 868 | 7.0% | 100.0% |
| NEG | 19 | 3 | 874 | 561 | 15.5% | 100.0% |
| Met | 19 | 5 | 1143 | 668 | 11.0% | 100.0% |
SUP
Supervised model analysis
Exploratory supervised-model standard. Models use all filtered features from the same complete 19 biological samples as the unsupervised QC, with training-fold mean centering and Pareto scaling. Overall PLS-DA uses two components. Each OPLS-DA uses one predictive and one orthogonal component for a prespecified binary comparison. Q2 and classification accuracy are calculated by four-fold stratified cross-validation; empirical P values use 200 label permutations. The genotype × fear interaction remains in the factorial univariate model and is not recast as binary OPLS-DA. Because n=4–5 per group, separation without positive Q2 and permutation support must not be treated as validated biology.
Lipidomics - positive ion mode
One four-group PLS-DA and four prespecified pairwise OPLS-DA models. Every card reports four-fold cross-validation and 200 response-label permutations.
Four-group PLS-DA
R2Y=0.434 · Q2=-0.011 · CV accuracy=0.42 · permutation P=0.060
OPLS-DA · FLOX-No fear vs FLOX-Fear
R2Y=0.982 · Q2=0.578 · CV accuracy=0.80 · permutation P=0.015
OPLS-DA · KO-No fear vs KO-Fear
R2Y=0.878 · Q2=0.189 · CV accuracy=0.67 · permutation P=0.124
OPLS-DA · FLOX-No fear vs KO-No fear
R2Y=0.885 · Q2=-0.455 · CV accuracy=0.33 · permutation P=0.622
OPLS-DA · FLOX-Fear vs KO-Fear
R2Y=0.958 · Q2=0.036 · CV accuracy=0.80 · permutation P=0.234
Lipidomics - negative ion mode
One four-group PLS-DA and four prespecified pairwise OPLS-DA models. Every card reports four-fold cross-validation and 200 response-label permutations.
Four-group PLS-DA
R2Y=0.440 · Q2=-0.208 · CV accuracy=0.21 · permutation P=0.433
OPLS-DA · FLOX-No fear vs FLOX-Fear
R2Y=0.978 · Q2=0.059 · CV accuracy=0.60 · permutation P=0.313
OPLS-DA · KO-No fear vs KO-Fear
R2Y=0.938 · Q2=0.006 · CV accuracy=0.67 · permutation P=0.294
OPLS-DA · FLOX-No fear vs KO-No fear
R2Y=0.958 · Q2=-0.479 · CV accuracy=0.44 · permutation P=0.697
OPLS-DA · FLOX-Fear vs KO-Fear
R2Y=0.985 · Q2=-0.465 · CV accuracy=0.40 · permutation P=0.682
Metabolomics
One four-group PLS-DA and four prespecified pairwise OPLS-DA models. Every card reports four-fold cross-validation and 200 response-label permutations.
Four-group PLS-DA
R2Y=0.408 · Q2=-0.265 · CV accuracy=0.21 · permutation P=0.463
OPLS-DA · FLOX-No fear vs FLOX-Fear
R2Y=0.915 · Q2=-2.075 · CV accuracy=0.10 · permutation P=0.970
OPLS-DA · KO-No fear vs KO-Fear
R2Y=0.907 · Q2=-0.049 · CV accuracy=0.56 · permutation P=0.264
OPLS-DA · FLOX-No fear vs KO-No fear
R2Y=0.854 · Q2=-1.227 · CV accuracy=0.44 · permutation P=0.811
OPLS-DA · FLOX-Fear vs KO-Fear
R2Y=0.874 · Q2=0.144 · CV accuracy=0.70 · permutation P=0.174
