The figure above briefly outlines the steps that are taken to gather data and prepare it for use in Gemma. Data are downloaded and imported into Gemma’s database. Currently Gemma supports data uploads from GEO and ArrayExpress (the latter with some restrictions) or from ad hoc sources in a tab-delimited format. In addition to the expression values, data loaders also attempt to import annotations and meta-data about the study where possible. The ‘array design’ (describing the transcript entities that were assayed), the experimental data and the experimental design (describing the conditions the samples represent, such as “tumor” vs. “control”) are then processed. The array design is subjected to sequence analysis and gene assignment. The experimental data is checked for quality and the experimental design provided with a structured description. The data are then analyzed and the results are stored in the system for later retrieval. Some of these steps are discussed in more detail in the following sections.
Gemma imports GEO series (GSE*), and obtains annotations from the associated dataset(s) (GDS*) if available. Along with these, the ‘platform’ (array design or microarray type, GPL*) is also imported. If data is imported from ArrayExpress, we reuse the GEO-originated platform if possible. It is common for a GEO experiment to use more than one platform. The data for the different platforms is combined in Gemma. The way this is done depends on how the experiment was designed. In some cases, the same samples were each run on more than one platform. In this case, the platforms are “stacked” so each sample’s expression vector includes the elements (probes) from all the platforms. In other cases, the samples were each run on just one platform, but the platform varied for sample to sample. For example, in some studies the Affymetrix HGU133A platform was used for some samples, and for other the Plus 2 array was used. The data for the elements in common are combined in Gemma, so the “stacking” is at the probe/sequence level.
Note that unlike the situation in GEO, a sample can only appear in one Gemma experiment (based on the “GSM” identifier). This means that some experiments imported from GEO do not have the same samples as the corresponding GEO entry, because samples that appear in experiments already in Gemma are removed at load time. For example, in GEO, ‘series’ are sometimes assembled into ‘super-series’; similarly, in GEO some samples appear in multiple overlapping studies. We also tend to use the super-series if the super-series is sufficiently cohesive (in our opinion), but in other cases we import only the constituent series independently.
Each expression platform (i.e. microarray type or “array design”) was re-annotated at the sequence level using methods essentially as described in Barnes et al. 2005. Briefly, the sequences were aligned to the appropriate genome assembly using BLAT. Repeatmasker was used to mask aligned regions containing repeats. The UCSC GoldenPath genome annotation database was then used to map high-quality alignments to genes based on the “known gene” and “Refseq” tracks, with the use of additional information from other tracks. Gene information including Gene Ontology annotation was imported from NCBI’s gene database with additional data from GoldenPath. Because our methods require sequence-level analysis (for mouse, human and rat), platforms which lack probe level sequence information are currently not usable for analysis in Gemma. Currently platforms for other organisms are annotated using manufacturer-provided information,. All the tools for interfacing with sequence analysis resources are provided as part of the Gemma source code distribution.
In addition to the free text imported along with the data, we are manually curating the data in Gemma to provide high-quality annotations. By ‘annotation’ we refer primarily to descriptive terms applied to each data set, and to the detailed description of the experimental design. Experimental designs are available on import of data from GEO in some cases (as “GEO subsets”). In all other cases, a curator uses the Gemma administrative tools to label each sample according to factors such as tissue and treatment. Experiments themselves are ‘tagged’ with terms describing the study at a higher level. For both types of annotations, as much as is practical we use terms from the MGED Ontology and other Open Biomedical Ontologies including the human disease ontology , the foundational model of anatomy, the chemical entities of biological interest and NIFSTD. Among the benefits of using ontologies are more uniformity within Gemma and better interoperation with other resources. At this writing, nearly all data sets in Gemma have experimental design details as well as broad annotations at the data set level. These annotations are used to help locate data sets in searches, as well as in performing statistical analyses.
To find out more about how you can edit the experimental design of your own data, please visit our Editing Experimental Designs page.
Although the data are presumably quality-controlled by the original publishers of the data sets prior to publication or submission to public repositories, we have undertaken our own quality control checks of the data. Therefore Gemma includes the ability to flag samples as outliers. The data from outliers are treated as missing by Gemma. Data sets with identified quality problems or other issues preventing preprocessing are retained in the system but ignored during analytical procedures.
Quality control in Gemma is both at the level of the data set and the level of annotations. First, we have examined each data set using diagnostic plots. We visualize the correlation between samples, which can be used to identify potential outliers.
Outliers are only flagged after a more detailed review, which includes referring back to the original publication and in some cases contacting the original authors for confirmation. We also visualize the distribution of correlations between probes, which is relevant to coexpression analysis. Datasets with “anomalous” correlation distributions (showing strong skew) are flagged. At the level of annotations, most data sets have been reviewed by at least two curators to ensure that experimental designs and other aspects of the experiment are described accurately and to help ensure uniformity and coherence of annotations across studies.
The list of experiments with removed outliers can be found here: http://chibi.ubc.ca/Gemma/expressionExperimentsWithQC.html
Another quality control step is looking at possible batch effects. The process is as follows:
- Download ‘raw’ data files from GEO
- Try to extract scan dates inside raw data files
- Group dates into batches
- Compare Principal Components to batches
- Compare batches to factors (confounds)
New in Version 1.8, data sets are now batch-corrected if possible, and Affymetrix are reanalyzed from CEL files where possible.
For more details on our differential expression analysis and coexpression analysis please see the Analysis Details page.