1. Applied Bioinformatics Week 9 Jens Allmer

2. Theory I Gene Expression Microarray

3. Gene Expression Is there a transcript? How much transcript is made? Is there any difference to the DNA? Is there any difference to the annotation?

4. Measure Expression Northern/Western Blot qPCR Next generation sequencing Microarray

5. http://www.nature.com/leu/journal/v17/n7/images/2402974f1.jpg

6. Chip Construction http://www.dkfz.de/gpcf/fileadmin/_migrated/RTE/RTEmagicC_AffyChipProduction.jpg.jpg https://www.bcm.edu/cms_web/110/affy3.jpg

7. http://www.scq.ubc.ca/wp-content/GeneChip.gif Chip Construction

8. http://angerer.swissbrain.org/expression_oveview.gif

9. http://www.scq.ubc.ca/wp-content/cDNAarray.gif

10. Bioinformatics Analysis Experimental design Standardization Data Analysis –Image processing, normalization –... –Clustering, Visualization Data Storage

11. List of MA Data Sources

12. A public repository for the archiving and distribution of gene expression data submitted by the scientific community. MIAME compliant data. Minimum Information About a Microarray Experiment http://www.mged.org/Workgroups/MIAME/miame.html http://www.mged.org/Workgroups/MIAME/miame.html Convenient for deposition of gene expression data, as required by funding agencies and journals. Curated, online resource for gene expression data browsing, query, analysis and retrieval. Gene Expression Omnibus (GEO): Gene Expression and Molecular Abundance Data Repository GEO Gene Expression Omnibus - TeachLine

13. GEO Architecture Platform (GPL) = the technology used and the features detected. Sample (GSM) = preparation and description of the sample. Series (GSE) defines a set of samples and how they are related. DataSets (GDS) sample data collections assembled by GEO staff. GEO has four kinds of data records Submitters may provide raw data Original microarray scans Raw quantification data GEO Gene Expression Omnibus - TeachLine

14. GPL Platform descriptions GSM Raw/processed spot intensities from a single slide/chip GSE Grouping of slide/chip data “a single experiment” GDS Grouping of experiments Curated by NCBI Submitted by Experimentalists Submitted by Manufacturer* GEO Architecture GEO Gene Expression Omnibus - TeachLine

15. GEO Home Page Simple interface to:  show status  find documentation  query data  browse data  submit data

16. Basic Search: Repository Browser Selecting the total public data or Repository Browser links on the GEO home page, takes you to the Repository Browser, listing: number of each type of submitted file, both public and unreleased the total number of each technology type under Platforms the total number of each Sample type

17. Basic Search: Browse Platforms All GEO submissions need to be associated with a platform file. These describe the features on a given platform, required to understand the data. A platform file must be submitted if one is not already present in GEO. Commercial array platform files are submitted to GEO by the manufacturer. GEO Gene Expression Omnibus - TeachLine

18. Basic Search: Browse Platforms Accession: GEO ID Title: brief description of platform Contact: submitter Samples: number of samples in GEO associated with platform ID Technology: platform type Release date: when file is publicly accessible The table can be sorted on any field except organism by clicking on the header. Specific platform files can be found using the ‘Find Platform’ option. GEO Gene Expression Omnibus - TeachLine

19. Basic Search: Find Platforms Select ‘Find Platform’ Select company Select distribution Select species Enter title keyword GEO Gene Expression Omnibus - TeachLine

20. Basic Search: Find Platforms (continued) Start the platform search Select the accession for the U133 plus 2.0 array Scroll down to find data table information GEO Gene Expression Omnibus - TeachLine

21. Data Retrieval: Browse Series Data is submitted to GEO as a Series, which represents the experiment design. Selecting Browse>Series brings up a list sorted by release date. Selecting a Series ID brings up the Series file summary. GEO Gene Expression Omnibus - TeachLine

22. Data Retrieval: Series Accession Page GEO Gene Expression Omnibus - TeachLine

23. GEO Accession Results Display Options Scope controls what information is displayed: Self Platform, Samples or Series Family Format controls how information is displayed: HTML SOFT (Simple Omnibus Format in Text) MINiML (MIAME Notation in Markup Language) Amount controls how much information is displayed: BriefQuick FullData All GEO accession results pages have the same header that allows different views and formats for the data to be displayed GEO Gene Expression Omnibus - TeachLine

24. Data Retrieval: Series Accession Page Biological sample summary Design summary Publication information Platform (total) Samples (total) GEO Gene Expression Omnibus - TeachLine

25. Data Retrieval: Sample File Summary Sample preparation Hybridization and data processing Platform Series GEO Gene Expression Omnibus - TeachLine

26. Data Retrieval: Sample File Data Table Data table field descriptions Truncated data table from Quick view Total data rows and file size Supplementary raw data file GEO Gene Expression Omnibus - TeachLine

27. Querying GEO with IDs from Papers A common way to access GEO data is through accessions from papers. Online journals include hyperlinks to the GEO accession page. Or, at the GEO home page enter the accession into the Query>GEO accession text box GEO Gene Expression Omnibus - TeachLine

28. GEO Links in PubMed Search Results One option for displaying PubMed search results is GEO DataSet links. When present, the results page is actually from Entrez GEO DataSets. GEO Gene Expression Omnibus - TeachLine

29. Advanced Searches GEO data can be queried as: Datasets: experiment-centric view using Entrez GEO DataSets Gene profiles: gene-centric view using Entrez GEO Profiles Selecting either takes you to a similar Entrez introduction page GEO Gene Expression Omnibus - TeachLine

30. Querying GEO DataSets Start a GEO DataSets search with the Query>DataSets text box This brings up an Entrez GEO DataSets results form Total results Number of DataSets Number of Platforms Number of Series GEO Gene Expression Omnibus - TeachLine

31. DataSet Search Result DataSet ID Description Platform Reference Series Supplementary files Number of Samples and truncated list Cluster image Select the DataSet ID or click on the cluster image to go to the DataSet record. GEO Gene Expression Omnibus - TeachLine

32. GEO DataSet Record Experiment design and DataSet information. Sample and analysis information. Data retrieval. Selecting analysis takes you to the data clustering interface. Selecting the cluster image takes you to the clustering page GEO Gene Expression Omnibus - TeachLine

33. GEO Gene Profiles GEO DataSet ID Platform ID, Platform Feature ID Gene description Target sequence accession Expression profile GEO Gene Profiles use gene IDs from Platform files to show the expression of a gene across DataSets. Entering a gene ID into the Query>Gene profiles text box takes you to the Entrez results page. GEO Gene Expression Omnibus - TeachLine

34. GEO BLAST On the GEO BLAST page enter sequences in fasta format, GenBank accessions or select sequence files on local disks for blastn comparisons. These are compared to GenBank sequences listed in Platform files associated with GEO DataSets From the Blast result page select the ‘E’ option to the right of an alignment to show GEO Gene Profiles for that sequence in GEO DataSets E button GEO Gene Expression Omnibus - TeachLine

35. End Theory I Mindmapping 10 min break

36. Practice I Gene Expression Omnibus –http://www.ncbi.nlm.nih.gov/geo/

37. NCBI GEO Take 15 minutes to browse the website

38. Repository Go to the repository browser –http://www.ncbi.nlm.nih.gov/geo/summary/ Explore the available tabs What kind of different data is available?

39. Where is the actual data? Try to find the following accessions: –GSE48874 –GSM1186226

41. End Practice I 15 min break

42. Theory II Next generation sequencing

43. Microarray vs NGS

44. Doug Brutlag 2011 The Human Genome How fast is the cost going down? 2006: $ 50 million 2008: $500,000 2009: $50,000 2010: $20,000 2011: $5,000 2012:??? $1,000 Thanks to Serafim Batzoglou

46. Roche/454 FLX: 2004 Illumina Solexa Genome Analyzer: 2006 Applied Biosystems SOLiD TM System: 2007 Helicos Heliscope TM : recently available Pacific Biosciencies SMRT: launching 2010 And many more

47. Doug Brutlag 2011 Illumina Solexa Sequencing Technology

49. Doug Brutlag 2011 Pacific Biosciences Sequencing

50. Doug Brutlag 2011 Phospholinked Fluorophores

51. Doug Brutlag 2011 Processive Synthesis

52. Applications of next-generation sequencing Jay Shendure & Hanlee Ji, Nature Biotechnology 26, 1135 - 1145 (2008)

54. OK, but where is the data? http://www.1000genomes.org/ http://trace.ddbj.nig.ac.jp/dra/index_e.html http://www.ebi.ac.uk/ena http://www.ncbi.nlm.nih.gov/Traces/sra/sra.c gi?view=studies http://www.ncbi.nlm.nih.gov/Traces/sra/sra.c gi?view=studies

55. End Theory II Mindmapping 10 min break

56. Practice I

57. NCBI http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi Browse the webpage for 15 minutes

58. Available Data Search for human data How much data is available? Find accession ERX628533 How large is the dataset? Why is it so large?

59. End Practice II

60. Homework Select 1 next generation sequencing platform and give a step by step description how it works Max 500 words and at most 5 figures.

62. http://dx.doi.org/10.6084/m9.figshare.100940

63. http://www.nature.com/nbt/journal/v26/n10/fig_tab/nbt1486_F1.html

64. http://www.nature.com/nbt/journal/v26/n10/fig_tab/nbt1486_F2.html

65. http://www.nature.com/nbt/journal/v26/n10/fig_tab/nbt1486_F3.html