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Molecular Profiling Colloqium
Janos DemeterDecember 15, 2006
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
• 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: controlH: HumanT: transgenesV: viral/bact
A: alternative exon - antisenseD: doping controlE: EST-derived oligoN: negative controlT: tiling
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
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
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
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
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)
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
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
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
HEEBO/MEEBO arrays in SMD:Quality control graphs
•Help page: http://smd.stanford.edu/help/arrayQuality.shtml
•Slides from tutorial: http://smd.stanford.edu/help/TUTORIALS/Repository.htm
• 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
• 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
• 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
• 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
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.
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
HEEBO/MEEBO arrays in SMD:Synthetic gene tool
• Pcl file sorted by name column• synthetic gene tool only looks at the first column
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
HEEBO/MEEBO arrays in SMD:Synthetic gene tool
The default option averages the rows and removes the original annotations
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 )
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
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
HEEBO/MEEBO arrays in SMD:Merge PCL files
Pcl1
Pcl2
Translation file
Combined PCL
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.
• 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
• 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