GPX-Macrophage Expression Atlas Help

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Introduction

The GPX Macrophage Expression Atlas (GPX-MEA) is a publicly available online resource for expression based studies of a range of macrophage cell types following treatment with pathogens and immune modulators. The GPX-MEA database is subdivided into three main components: Experiments, the complete set of minimum information about a microarray experiment (MIAME) information including the raw data files, Arrays, which refers to information about the design and manufacture of the array itself, and Probes, which provides information on the individual array reporter elements (Probes) and their associated measurements.

Searching the database

GPX-MEA provides three main interfaces for querying the database, Experiments, Arrays and Probes. Each query interface provides a number of search fields by which data within GPX can be searched and retrieved. The query interfaces can be accessed via the three tags (Experiments, Arrays and Probes) from the GPX-MEA navigation bar.

GPX-MEA navigation bar

GPX-MEA navigation bar

 

Experiments

To find an experiment of interest within the database the user can use any of the search fields within the Experiments search interface. For example, each experiment record has an accession number in the format GPX-NNNNN.N, (where N is a number). This accession number may be used to quickly identify specific experiments in the database. Alternatively, there are a variety of other search fields provided that enable the user to search and retrieve data. A screenshot of the experiment query interface is shown below.

Note: Each search field is mutually exclusive and cannot be used in combination with other search fields.

Search experiment interface
experiment search interface

Following a search the results for a given search are displayed at the foot of the interface and contain hyperlinks to associated experiment records. It is also possible to view all experiment records in the database by selecting the All experiments link.

Experiment Record

The experiment record contains all the information associated with an experiment. The experiment record is divided into six sections. Experiment details, general experiment information. Experiment Protocols, the hybridisation, scanning and Image Analysis Protocol. Measurements, a table that contains source, sample, sample treatment, extraction protocol, labelling protocol, image analysis files and measurements. Array Design, conatins link to the Array record. Quality Assessment, the quality assessment score and a link to a detailed breakdown of the score. To view details about a specific protocol, sample, array design or the Quality assessment select the associated link.

Experiment record

experiment record

Arrays

To view a list of all the array types stored within the database select the Arrays tab on the navigation bar. This will display a list of all the array types stored within the database and contains links to the full array record.

Probes

To search for probe information, select the Probes tab on the navigation bar. From the Probes interface you can search for probes using any of the seach fields. From the results of a search you can navigate to the probe record by selecting Show Details link.

Probe search interface

probe select

Note- Gene Symbol Searches: Searching using the Gene Symbol fields will first try to find a match using the Official Gene Symbol. If a match is not found, GPX-MEA will search using the submitted term as a synonym or gene name. If a match is found a list of potential gene matches will be presented. In the example below the user searches with the term psd95 in the Official Gene Symbol field. GPX-MEA does not recognise psd95 in its list of official gene symbols and searches psd95 against it lists of gene synonyms. Three synonym matches are found and GPX-MEA presents a list of potential matches with their associated official gene symbol. The user then conduct the search with the official gene symbol using the Search link.

Results from a psd95 search

synonymns_results

 

Probe Record

Selecting the Show details link from a probe search result navigates the user to the Probe record interface. The probe record contains probe annotation and links to the Affymetrix NetAffx , NCBI Gene and Adapt websites. In second section isplays the experiments within GPX-MEA that contain measurements for that probe. The third section displays graphs of the probes expression for each sample in a given experiment. Expression levels are TGT-100 normalised. The graph bars are colour coded to indicate the Affymetrix present(red), marginal(blue) or absent(green) call.

Probe record

probe record

View Probe Expression Levels

To view the expression level of a particluar probe navigate to a Probe record using the Probes search interface. From the Probe record check the experiments that you wish to view expression values from and select the Get Expression Levels button.

Probe expression levels results

expression levels

The results report the normalised signal intensity (TGT 100), the Detection call and the Detection significance from each sample in the choosen experiments. In addition, hyperlinks to the experiment and sample record are provided.

Measurements and Data files

GPX-MEA stores measurement data for each experiment, A measurement is total gene expression content for a single array. All measurement data from Affymetrix GeneChip experiments have undergone TGT-100 normalization. TGT-100 values are recalculated from the original image analysis files (CEL) files using Affytmetrix Gene Chip Operating Software. Other values stored within the database are the associated detection call (P, M, A) and the p-value of detection. A comma separated values (CSV) file for each measurement can be downloaded from the experiment record for further analysis.

Downloading Measurements

To download the measurements from an experiment navigate to an experiment record. From the experiment record go to the measurements sections. Check the measurements you wish to download in the Measurement Id column. Alternatively, press the Check All button to select all measurements for download. To download the checked measurements press the Get Measurements(s) button. Measurements are returned to the user as Comma Separated Value (CSV) files.

Downloading Files

To download Image Analysis files(CEL) navigate to an experiment record. From the experiment record go to the Measurements section. From the Measurements section select the link to the Image Analysis files from the Image Analysis File column. Note: Affymetrix CEL files available for download are in binary format (Version 4.0). Only one image file maybe downloaded at any one time.

Quality Assesment Score

A quality control assessment is made for each experiment curated into GPX-MEA. A detailed description of the quality assessment score can be viewed from a link in the Quality Assessment section in the Experiment Record.The quality assessment score is calculated using quantitative and qualitative scores in different areas: use of Standard Operating Procedures, the number and use of independent biological replicates, quality of image, sample quality control and structure of numeric data (spread, reproducibility). The total score ifor each section is calculated by multiplying the weight of the assessment category by the score of the criteria. For eaxmple, An experiment with three biological repliactes per group is scored as 1 in the scoring criteria and multipled by the weight of the category, 3, to give a total of 3. A combination of the different categories is used to give a total quality assessment score. This score can be used to indicate the quality of the underlying experiment in the database and as such is subject to interpretation. Where data is unavailable for assessment no score is assigned.

Quality assessment criteria for GPX-MEA

Assessment category

Tool

Weight

(Score)Criteria

Standard Operating Procedures (SOPs)

Experiment description/protocol

2

2 – Standard Operating Procedures have been documented and followed.

1 – Only partial SOPs have been documented applied or followed

0 – SOPs not known or not followed

Sample size

Experiment description/protocol

3

2 – Number of independent biological samples exceeds 5 per group, allowing for reasonably high statistical power in the hypothesis testing.

1- 3-5 independent biological samples per group, allowing to apply minimum level of statistics

0 - 1 or 2 independent biological samples per group (e.g. pilot experiment), reducing the applicability of statistics.

Image quality

Image scan or numerical representation (Affymetrix)

3

2 – No signs of chip defects, background noise (RawQ) within 5 point range of each other. Scaling factor within 1-2 fold range.

1 – Isolated chip defects, RawQ within 5 point range, scaling factor 1-3

0 – Systematic chip defects, saturation, RawQ outside 5 point range, scaling factor greater than 3.

Target sample quality

Electropherogram, UV-Spectrophotometer

1

2 – Presence of 18S and 28S ribosomal subunits with no sign of degradation in any sample (RNA integrity). Absorbance ratio of A260/280 close to 2 (RNA purity) (15) .

1 – Either RNA integrity or purity criteria are met.

0 – Neither integrity nor purity criteria are met, or sample quality assessment was not performed, or no information provided.

Sample level data variation/distribution

Box-and-whisker plots, supporting MA plots if required.

3

2 – Very consistent array medians and Inter-Quartile-Ranges across all samples before normalisation procedures (note: underlying assumption is that any treatments/conditions should not cause differential expression in more than 5-10% of all gene probes on the array.)

1 – Very small number of inconsistent data distributions with assumptions of a correctable difference in array signal intensity met (all genes on array affected, inter-array gene relationships are linear or global differences are expected, e.g. LPS treatment).

0 – Inconsistent median signal levels and spread (IQR) of data throughout the experiment, no biological explanation given.

Gene level data variation

Coefficient of Variation versus Mean Expression plots

2

2 – Coefficient of Variation for majority of genes across all replicates within a sample group is less than 20%. Genes above this CV level are mostly in the lower signal range (due to measurement error).

1 – A small proportion of all genes has higher CV values than 20%, with a small number of these in the medium to high expression range.

0 – A large proportion of genes has a CV above 20% across most of the expression range.

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