1. URL HTTP://WWW.INSILICOGEN.COM PHONE 031.278.0061 FAX 031.278.0062 E-MAIL INFO@INSILICOGEN.COM ADDRESS #909, VENTURE VALLEY, 958, GOSAEK-DONG, GWONSEON-GU,SUWON, GYEONGGI-DO, KOREA 441-813 Copyright ⓒ Insilicogen, Inc. 2009. All rights reserved. Tutorial: Expression analysis part Ⅰ ~ Ⅳ 2009 – 03- 04 김 경 의

2. Importing array data NCBI Gene Expression Omnibus(GEO) database 에서 data set download :http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE6943 &targ=gsm&form=text&view=data 데이터 다운로드 후 원하는 디렉토리에 저장하고 Toolbar 에서 파일을 Import 한다. ^SAMPLE = GSM160089 #ID_REF = #VALUE = GCOS signal #ABS_CALL = Present/absent per Affy software !sample_table_begin ID_REFVALUEABS_CALL

3. Import Annotation file Affymetrix web site: http://www.affymetrix.comhttp://www.affymetrix.com RAE230A 를 검색하여 annotation file 을 다운로드합니다.

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

5. Defining the number of groups

6. Group 을 Delete 할 수 있고, Add New Group 을 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. 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 =log ₂ R - log ₂ G A : mean log-intensity = (log ₂ R + log ₂ G)/2 M 과 A 값을 이용한 Plotting 은 위의 log ₂ R 과 log ₂ G 를 이용한 plot 을 45° 회전시킨 plot 으로 0 값 을 기준선으로 gene data 를 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. Group differentiation Toolbox | Expression Analysis | Quality Control | Principal Component Analysis

23. Principal component analysis colored by group

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

25. 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

26. Sample clustering

27. Result of hierarchical clustering of samples Show Heat Map

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

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

30. Hierarchical clustering of features

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

32. Statistical analysis – ANOVA Two groups 이상 선택했을 경우

33. Corrected p-values

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

35. Filtering on FDR p-values

36. Inspecting the volcano plot Ctrl key 를 누르고 volcano plot 을 누르면 두개의 view 가 나타난다. 선택 된 데이터에 대해서는 dot 이 붉은색으로 표현된다.

37. Filtering absent/present calls and fold change Add search criterion (+) button 을 누르면 criteria 를 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

38. Saving the gene list

39. New experiment Save

40. Processes that are over-represented in the small list Toolbox | Expression Analysis | Annotation Test | Hypergeometric Tests on Annotations

41. The result of testing on GO biological process

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

43. Gene set enrichment analysis based on GO biological process

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

45. Add Array Annotations Toolbox | Annotations test | Add Array Annotations

46. Download Sequence Select 한 개수 만큼 sequence 를 download 할 수 있습니다.

47. Created sequence 선택한 개수만큼 sequence 생성

48. Saving sequence Sequence name 을 하나씩 드래그하여 Navigation Area 에 저장합니다.

49. BLAST Search Toolbox | BLAST Search | NCBI BLAST 방금 저장한 sequence 를 선택 3 개의 sequence 를 한번에 BLAST Search 할 수 있음

50. Choose program and database

51. BLAST Search result