1. Molecular Profiling Colloqium Janos Demeter December 15, 2006

2. HEEBO/MEEBO arrays in SMD Entering doping control data into SMD Quality control graphs Synthetic gene tool to compare data from cDNA and HEEBO/MEEBO arrays Merge pcl files tool Current state of annotation

3. Source:http://alizadehlab.stanford.edu Sequence_id: hSQnnnnnn -In SMD: cloneid meaningless, but unique to a given oligo sequence Oligo_id: hXXnnnnnn unique to a well, not a sequence ) -in SMD: oligo_id the XX codes have meaning: HEEBO/MEEBO arrays in SMD: Nomenclature C: control H: Human T: transgenes V: viral/bact A: alternative exon - antisense D: doping control E: EST-derived oligo N: negative control T: tiling

4. HEEBO/MEEBO arrays in SMD: Connect to SOURCE with oligo/seqID Annotations of heebo/meebo oligos can be retrieved from SOURCE by linking to: http://source/cgi-bin/source/sourceRedirectSeqID.pl?seqID=hSQ000011 Or: http://source/cgi-bin/source/sourceRedirectSeqID.pl?oligoID=hHC007895

5. HEEBO/MEEBO arrays in SMD: Entering doping control data Heebo/meebo arrays contain a lot of various controls To take advantage of the doping controls, it is essential to know the amounts that were added to your samples SFGF tells you how much is in 1 microliter of doping control mix, but amplification/ dilution might change that SMD needs to know how much you add in the sample compared to how much SFGF tells you to add Added problem: 4 tubes from SFGF: MJ and Ambion_Stratagene, Cy3 and Cy5

6. HEEBO/MEEBO arrays in SMD: Entering doping control data Experiment entry form can capture all this DCV2.1 = DCV2.1_Ambion_Stratagene + DCV2.1_MJ If no amplification, follow SFGF suggestion, enter: DCV2.1, factor1=1, factor2=1 If amplified/diluted controls, enter values for each tube: DCV2.1_MJ, factor1=1.5, factor2=1.6 DCV2.1_A_S, factor1=1.932, factor2=0.8

7. Heebo/meebo arrays in SMD: Entering doping control data Experiment entry form can capture all this DCV2.1 = DCV2.1_Ambion_Stratagene + DCV2.1_MJ If no amplification, follow SFGF suggestion, enter: DCV2.1, factor1=1, factor2=1 If amplified/diluted controls, enter values for each tube: DCV2.1_MJ, factor1=1.5, factor2=1.6 DCV2.1_A_S, factor1=1.932, factor2=0.8

8. HEEBO/MEEBO arrays in SMD: Quality control graphs HEEBO/MEEBO quality assessment graphs from BioConductor package (Agnes Paquet/UCSF) Per array graphs that use doping, tiling mismatch and negative controls For batch/uploaded gpr files: can be reached from main page For individual expts: from data display page For new expts with doping control: graphs are automatically created at data loading The last set of graphs are available from view expt page New set of graphs Previously created set (or during data loading)

9. HEEBO/MEEBO arrays in SMD: Quality control graphs Can be used for a gpr file uploaded from desktop - print has to be present in SMD and oligo_ids in the id/name column In batch for a result set list on loader.stanford.edu If called for a specific experiment, the values are already filled in. Normalization options available from limma. Note that this will NOT change your data in SMD, but is only used to generate the graphs Background subtraction methods - same story as normalization Job is placed in the job-queue - email is sent with link

10. HEEBO/MEEBO arrays in SMD : Quality control graphs Can be used for a gpr file uploaded from desktop - print has to be present in SMD and oligo_ids in the id/name column In batch for a result set list on loader.stanford.edu If called for a specific experiment, the values are already filled in. Normalization options available from limma. Note that this will NOT change your data in SMD, but is only used to generate the graphs Background subtraction methods - same story as normalization Job is placed in the job-queue - email is sent with link

11. HEEBO/MEEBO arrays in SMD: Quality control graphs Can be used for a gpr file uploaded from desktop - print has to be present in SMD and oligo_ids in the id/name column In batch for a result set list on loader.stanford.edu If called for a specific experiment, the values are already filled in. Normalization options available from limma. Note that this will NOT change your data in SMD, but is only used to generate the graphs Background subtraction methods - same story as normalization Job enqueued in the job-queue - email is sent with link

12. HEEBO/MEEBO arrays in SMD: Quality control graphs Help page: http://smd.stanford.edu/help/arrayQuality.shtml http://smd.stanford.edu/help/arrayQuality.shtml Slides from tutorial: http://smd.stanford.edu/help/TUTORIALS/Repository.htm http://smd.stanford.edu/help/TUTORIALS/Repository.htm

13. MA-plots before and after normalization A = 1/2*(log2(Cy5) + log2(Cy3)) M = log2(Cy5 / Cy3) Loess lines are shown for sectors if print-tip normalization was selected Distribution should be centered around M=0, with no intensity dependence HEEBO/MEEBO arrays in SMD: Diagnostic graphs

14. Tiling probes were designed along the transcript: 17 human genes (actin - 6 … LRP1 - 89 oligos Non-normalized signal intensities (Cy5 and Cy3) vs. probe’s distance from 3’-end Quick drop in signal indicates problem in sample (degradation/ivt) HEEBO/MEEBO arrays in SMD: Tiling control graphs

15. Mismatch and tiling probes are used to test the degree of cross- hybridization among homologous probes Mutations are anchored (at the extremities) or distributed (along transcript) Calculated binding energies vs. normalized (i.e. divided by median of corresponding wild type probes) raw intensities HEEBO/MEEBO arrays in SMD: Mismatch control graphs

16. Observed vs. expected log-ratios (normalized and bg corrected) for each doping control group Ratios should be aligned on the diagonal Graphs for individual doping controls as well Shows the range where the log(mass ratio) vs. log(intensity ratio) is linear HEEBO/MEEBO arrays in SMD: Doping control graphs

17. HEEBO/MEEBO arrays in SMD: Synthetic gene tool There is a help page for using synthetic gene tool: http://smd.stanford.edu/help/synthGenes.shtml A "synthetic gene" is a group of "reporters" (clones, oligos, ORFs, etc.), together with some method of combining their expression vectors. Very useful tool, great flexibility in combining data rows. One use of it: compare data from various platforms, e.g. oligo to cDNA prints. Available from repository and applicable to a pcl file.

18. HEEBO/MEEBO arrays in SMD: Synthetic gene tool How to use it to compare heebo and cDNA arrays?: Select experiments from cDNA and heebo prints Selected biological annotation is not important for collapsing data What is important: include uid Save the pcl file in your repository

19. HEEBO/MEEBO arrays in SMD: Synthetic gene tool Pcl file sorted by name column synthetic gene tool only looks at the first column

20. HEEBO/MEEBO arrays in SMD: Synthetic gene tool To access the tool, click the “synth” icon in the repository Rows can be collapsed based on a number of prepared lists - now LocusLink should be selected The default option will remove the original ids and annotations and replace the rows with the average

21. HEEBO/MEEBO arrays in SMD: Synthetic gene tool The default option averages the rows and removes the original annotations

22. HEEBO/MEEBO arrays in SMD: Synthetic gene tool Collapse of rows by any arbitrary grouping of genes Prepared lists are available for - chromosomal locations - cytobands - locusid - clusterid - transcript length groups - cancer modules (E. Segal) - tissue types - processes - any other genelist in user’s genelist directory on loader Name of genelist will become the name of synthetic gene. Individual reporters can be weighted ( -1 to 1 )

23. HEEBO/MEEBO arrays in SMD: Synthetic gene tool Average rows (reporters) by synthetic gene and: - don’t remove original data rows - remove averaged data rows (but keep the ones that don’t belong to any synth gene) - remove all original data rows Don’t average, only annotate the rows with synthetic gene annotation (prepend name column): - keep/don’t keep original annotation

24. HEEBO/MEEBO arrays in SMD: Merge PCL files Combine two (or more) pcl files into single pcl file - files can be on the desktop or in repository In the process: - average (optionally) columns (experiments) with the same name - average (optionally) rows (genes) based on a translation file Averaging can be mean or median

25. HEEBO/MEEBO arrays in SMD: Merge PCL files Pcl1 Pcl2 Translation file Combined PCL

26. HEEBO/MEEBO arrays in SMD: State of annotation Meebo: anotation complete and is in SMD Heebo: anotation complete, but some oligo annotations are not in SMD yet. Annotations: geneid (locusid) gene name gene symbol chromosome location (in gff file) GB accession (RefSeq/est) Problem: ~500 oligos are annotated to more than one gene (~1000 spots involved) - these cases can’t be correctly represented in the database currently. The fields that have conflict are not entered into SMD.

27. For each sequence (sequence_id) we can have only one set of annotations. We have developed a new biosequence schema for SMD, to model the relationships between sequences, genes and genomes in a more biologically meaningful manner. Among other things, the new schema will allow us to map one sequence to more than one gene. We are currently migrating existing sequence annotations to tables using the new biosequence schema. Once this is finished (soon), all the biological annotations for the HEEBO arrays will be available in SMD. HEEBO/MEEBO arrays in SMD: State of annotation

28. Updates Genome coordinates: When a new genome version is released, the oligos need to be BLASTed anew (last time: spring of 2005, meebo: 2004) to find the coordinates of oligos. New releases have been made 1- 3 times a year. Result: oligos to chromosomal locations. Biological annotations: Annotations need to be updated to capture new knowledge. Result: chromosomal coordinates to genes. Currently, no updates are done for the sequences on the HEEBO/MEEBO arrays. They will be worked out after we have the new biosequence tables in place. HEEBO/MEEBO arrays in SMD: State of annotation