1. Tutorial: Expression analysis part Ⅰ~ Ⅳ
2009 –
김 경 의

2. Importing array data
NCBI Gene Expression Omnibus(GEO) database에서 data set download :
데이터 다운로드 후 원하는 디렉토리에
저장하고 Toolbar에서 파일을 Import한다.
^SAMPLE = GSM160089
#ID_REF =
#VALUE = GCOS signal
#ABS_CALL = Present/absent per Affy software
!sample_table_begin
ID_REF VALUE ABS_CALL

3. Import Annotation file
Affymetrix web site:
RAE230A를 검색하여 annotation file을 다운로드합니다.

4. Toolbox | Expression Analysis | Set up Experiment
Grouping the samples
Toolbox | Expression Analysis | Set up Experiment

5. Defining the number of groups

6. Defining the number of groups
Group을 Delete 할 수 있고, Add New Group을 이용하여
추가 할 수도 있음

7. Naming the groups

8. Assigning the samples to groups
First 6 samples right-click and select Heart,
Select the last 6 samples, right-click and select Diaphragm

9. The experiment table Total present count :
The number of present calls for all samples.
IQR-Expression values:
The interquartile range for all samples.

10. Annotation level

11. Add annotations, Create experiment, Download sequence
Add Array Annotations: Adding array annotations
Create Experiment from selection: creating a sub-experiment from a selection
Download Sequence: Downloading sequences from the experiment table

12. Toolbox | Expression Analysis | General Plots | Create MA Plot
Transformation
Toolbox | Expression Analysis | General Plots | Create MA Plot

13. Scatter plot view of an experiment
, Inside
, Major ticks
X axis
Y axis

14. MA plot before transformation
M : log-intensity ratio
=log₂R - log₂G
A : mean log-intensity
= (log₂R + log₂G)/2
M과 A값을 이용한 Plotting은 위의 log₂R
과 log₂G를 이용한 plot을 45° 회전시킨 plot으로 0값을 기준선으로 gene data를 관찰

15. Transformation
Toolbox | Expression Analysis | Transformation and Normalization | Transform

16. Normalization
Toolbox | Expression Analysis | Transformation and Normalization | Normalize
Select a number of samples or an experiment and click Next

17. Choose normalization method

18. Normalization settings

19. MA plot after transformation

20. Comparing spread and distribution
Toolbox | Expression Analysis | Quality Control | Create Box Plot

21. Box plot of the 12samples in the experiment

22. Toolbox | Expression Analysis | General Plots | Create Histogram

23. Selecting which values the histogram should be based on
Show Table

24. Table view of a histogram

25. Group differentiation
Toolbox | Expression Analysis | Quality Control | Principal Component Analysis

26. Principal component analysis colored by group

27. Dot properties | select GSM160090 in the drop-down box | Show names
Naming the outlier
Dot properties | select GSM in the drop-down box | Show names

28. Hierarchicla clustering
Toolbox | Expression Analysis | Quality Control | Hierarchical Clustering of Samples
Leave the parameters at their default and click Finish
Euclidean distance
1 – Pearson correlation
Manhattan distance
Single linkage
Average linkage
Complete linkage

29. Sample clustering

30. Result of hierarchical clustering of samples
Show Heat Map

31. Feature clustering
Toolbox | Expression Analysis | Feature Clustering | Hierarchical Clustering of Features

32. Parameters for hierarchical clustering of features
Euclidean distance
1 – Pearson correlation
Manhattan distance
Single linkage
Average linkage
Complete linkage

33. Hierarchical clustering of features

34. K-means/medoids clustering
Toolbox | Expression Analysis | Feature Clustering | K-means/medoids Clus-tering

35. Parameters for k-means/medoids clustering

36. Parameters for k-means/medoids clustering

37. Five clusters created by k-means/medoids clustering

38. Statistical analysis – T-tests
Toolbox | Expression Analysis | Statistical Analysis | Statistical Analysis

39. Statistical analysis – ANOVA
Two groups 이상 선택했을 경우

40. Corrected p-values

41. FDR p-values compared to Bonferroni-corrected p-values

42. Filtering on FDR p-values

43. Inspecting the volcano plot
Ctrl key를 누르고 volcano plot을 누르면 두개의 view가 나타난다.
선택 된 데이터에 대해서는 dot이 붉은색으로 표현된다.

44. Filtering absent/present calls and fold change
Add search criterion (+) button을 누르면 criteria를 추가할 수 있다.
Filtering genes where at least 5 out of 6 calls in each group are present.
The absolute value of group mean difference should be larger than 2

45. Saving the gene list

46. New experiment Save

47. Processes that are over-represented in the small list
Toolbox | Expression Analysis | Annotation Test | Hypergeometric Tests on Annotations
Highest IQR: the feature with the highest interquartile range(IQR) is kept
Highest value: the feature with the highest expression value is kept

48. The result of testing on GO biological process

49. Gene Set Enrichment Analysis (GSEA)
Toolbox | Expression Analysis | Annotation Test | Gene Set Enrichment Analysis(GSEA)
Original full experiment select

50. Gene set enrichment analysis based on GO biological process

51. The result of a gene set enrichment analysis based on GO biological process

52. Toolbox | Annotations test | Add Array Annotations

53. Download Sequence
Select 한 개수 만큼 sequence를
download 할 수 있습니다.

54. Created sequence
선택한 개수만큼 sequence 생성

55. Saving sequence
Sequence name을 하나씩 드래그하여 Navigation Area에 저장합니다.

56. Toolbox | BLAST Search | NCBI BLAST
방금 저장한 sequence를 선택
3개의 sequence를 한번에 BLAST Search 할 수 있음

57. Choose program and database

58. BLAST Search result