Q&A ToxProfiler

QA - ToxProfiler

• Q1. How many spleen samples of the example dataset are stored in Tox-Profiler?– 17

• Q2. What cell types were used in the study by Kevin Hochstenbach?– in vitro studies in peripheral blood mononuclear cells

• Q3. What type of microarray was used in the Spicker dataset?– GeneChip Rat230 version 2 (via GEO)

QA - ToxProfiler

• Q4. What compound was used in this example dataset? How many groups of samples are there in “Spleen HCB”, what do they represent and how many samples/group?– HCB = hexachlorobenzene– 16 samples in 3 groups: C=control (5), L=low dose (6)

and H=high dose (5)

• Q5. How many genes where measured on each array?– 4188

QA - ToxProfiler• Q6. Which gene is downregulated the most in C1 compared to

control and what is its z-score?– LOC287167 -2.61 Globin, Alpha

• Q7. Which gene is downregulated the most in H5 compared to control and what is its z-score?– MGP -9.16 Matrix Gla Protein

• Q8. Which array (C1 or H5) shows the strongest deviation from control? Can you confirm this based on the Std. Dev./Variance of the sub-experiments? Please give a biological explanation for this difference in deviation (keep it simple!). Assuming there are no biological differences, would you still expect a difference in deviation (think about how data was processed).– H5 (abs z-scores H5 > abs z-scores H1)– yes, .117 vs .399 (in right part table)– exposure to a high dose of HCB should make a difference!– we took average of C‘s as control and divided all samples with it, so C‘s

should have smallest ratios

QA - ToxProfiler

• Q9. Make a schematic drawing of this example situation in the case that the set of cell cycle genes are downregulated in H5. Do this by drawing normal distributions (bell shapes) and indicate in the drawing which characteristics of the distribution (shape) cause a strong negative t-score. – small std and large difference in means

background

cell-cycle

large

smallsmall

QA -ToxProfiler

• Q10. Which type of pathways are upregulated and which type of pathways are downregulated in H5?– cell cycle up– B/T signalling down

• Q11. Which pathways are not significantly up/down with the default threshold E-value at 5%, but would be if the E-value threshold would be less stringent? Are these still biologically relevant? – ATM, p53 (up) and IL signalling (down)– YES

QA - ToxProfiler• Q12. What is the average ratio of the “Cell cycle” genes in H5 and how many genes

(Orfs) are associated to the cell cycle pathway in WikiPathways?– 1.89– 28

• Q13. Describe the general relationship between exposure to the compound and the Cell Cycle (ignoring some deviations).

– increasing dose leads to increasing upregulation of cell cycle• Q14. Which sub-experiment(s) shows deviating behavior in terms of this relationship?

– L6 and H4 are lower than expected• Q15. How many Cell Cycle genes are downregulated in H5?

– 9• Q16. Compare the log2 values of the cell cycle genes in H5 with the sub-experiment

in the high dose that you found deviating in Q14. Explain the reason for the difference in T-score

– the strong upregulated genes in H4 are much less (only 4 >.5 vs 12)– downregulated is similar

QA - ToxProfiler• Q17. Which pathway, besides “Cell Cycle” is also upregulated in the

high dose HCB exposure group in Spleen?– DNA replication

• Q18. Are there any pathways clearly downregulated at high dose in Spleen? If so, which pathways and do they show a dose-response effect? If not, check which pathways come close and discuss a situation in which a pathway may not show up in the overview, but still might be biologically relevant.– No, T/B cell receptor signalling come close– L is not clearly effected, but H is– Some Hs are very close to threshold but not over it– if all H are very close to threshold

QA - ToxProfiler• Q19. Which BioCarta pathway is most strongly effected by the

compound?– Hemoglobin’s chaperone 11.88

• Q20. Which of the "CMGS - Tissue Genes Human" genesets has the highest/lowest T-Value for individual sub-experiments, BUT shows no dose response? At which sub-experiments does this geneset peak? Can you think of a biological/technical reason that explains this result?– Pancreas– peaks at C3, L4, H4– can this tissue be accidently added when preparing samples?

• Q21. Which “CMGS Signal Transduction” pathway responds particulary to low dose conditions?– IL3_up is up at low dose

QA - ToxProfiler• Q22. What colors are used in the heatmap to represent positive/high

and negative/low T-values?– red is positive, blue is negative, white is close to zero

• Q23. Which sub-experiments behave differently compared to the others in their group?– L2, H2, H3

• Q24. Describe the four main groups of pathways and describe their particular behavior in relation to the compound. (take a representative process per group and describe roughly up/down for C, L and H).– metabolism, oxidative stress: C=0,L=UP,H=UP– GPCR: C=mixed, L=up, H=0– IL6: down at C6– ox phos: particular down at L– fatty acid: down at L and H

QA - ToxProfilerQ25. Compare these findings to the “Results and

Discussion” part of the corresponding paper and describe at least one finding in agreement with the paper.

Additional Excersise 1

• some tissue-specific genesets are up in both organs