QC Chart Tool guide - Agilent Technologies

Agilent QC Tool (v1.0)

User Guide

QC Tool User Guide (v1.0)

Copyright 2006, Agilent Technologies

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Contents 1

Overview ........................................................................................................... 3

2

Objective ........................................................................................................... 3

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Installation ........................................................................................................ 4

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Loading Data .................................................................................................... 5

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Associate Annotations...................................................................................... 8 Export-All Method ............................................................................................. 8 Export-Subset Method ..................................................................................... 11 Generate-New Method ..................................................................................... 11

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Defining Queries............................................................................................. 11

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Defining Metric Sets and Thresholds ........................................................... 16 7.1 Defining Metric Sets ............................................................................. 17 7.2 Defining Metrics ................................................................................... 17 7.3 Set Thresholds....................................................................................... 23 7.4 Export and Import Metric Sets.............................................................. 25 7.5 Setting the Mandatory and In Range Evaluation status for metrics...... 26 7.6 Removing and duplicating the metric-sets............................................ 26 7.7 Removing metrics ................................................................................. 27

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Defining a QC Chart...................................................................................... 27 Color by In\Out of Range................................................................................. 28 ShapeBy and ColorBy options ......................................................................... 28 Viewing the QC Chart...................................................................................... 28 Using the QC Chart View Options .................................................................. 29

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Extraction Queries and QC Charts Pane .................................................... 30 Extraction Queries Pane................................................................................... 30 QC Charts Pane ................................................................................................ 31

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Extraction Results Pane................................................................................. 31

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Reset and Remove Database operations ...................................................... 32

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Application Themes ....................................................................................... 33

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Use of Metric sets in Feature Extraction...................................................... 33

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Overview of Feature Extraction & QC Chart Tool Connection................ 35

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Feature Extraction FEParameter fields used in QC Chart Tool............... 36



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1

Overview

The QC Chart tool is a desktop application that accumulates, aggregates and analyzes summary statistics from Feature Extraction output files and optional annotation files for the purpose of monitoring microarray processing performance. The data is housed in a relational database from which queries can be generated and saved to subset the data into user-defined logical groupings such as by experiment, by batch, or by date of processing. Using the data, metrics can be generated that monitor aspects of the microarray processing workflow. Metric sets can be formed that combine metrics that monitor different aspects of microarray processing. With metric sets, you can plot graphically the results from historical data and generate thresholds for the metrics that are appropriate for your experimental conditions and processing environment. The metric sets and thresholds can be used in the QC Chart tool on a regular basis to monitor processing performance. They can also be imported to Feature Extraction software to monitor each array and batch as it is processed. The QC Chart Tool is meant to be used in a production environment where: 1. Biological samples are similar 2. Microarray processing protocols are identical 3. Monitoring run-to-run consistency is an important goal The QC Chart Tool v1.0 is provided as an optional accessory to Feature Extraction (FE) 9.1. The software must be installed on a computer with FE 9.1 already functionally installed but does not require a separate license to operate. Agilent provides the software “AS IS” and any express or implied warranties, including, but not limited to any implied warranties of merchantability, satisfactory quality, reasonable care and skill, and fitness for a particular purpose are expressly disclaimed. For the most recent information on the QC Chart Tool, please go to the Web site: http://www.agilent.com/chem/FEQCMetrics This web site will contain updates to the software and this manual as well as example metric sets as they become available.

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Objective

The QC Chart utility provides the ability to determine the quality of extractions. Currently Feature Extraction software can run a batch of arrays generating a batch summary and a quality control report (QC Report) per microarray. In order to ensure the quality of the array, you should check the QC report of each array. To have a summary of quality of batch extractions, the QC Chart Tool is introduced. This tool will allow you to define quality metrics, apply those metric criteria to extraction data, and verify quality metrics through QC Charts.



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This tool has the following features (see Appendix 1 for overview): • Ability to have all extractions in a central repository • Ability to share central repository • Ability to annotate extractions with custom annotation. • Ability to filter extractions by defining queries • Define metrics to monitor quality • Define thresholds for metrics to check performance/quality of extraction • Define metric sets as a set of metrics (with or without thresholds) • Define a Quality chart (“QC Chart”) by defining query filters, metric set, and chart formatting on extraction set • Export QC Chart data for further use • Export metric set for use in FE • Evaluate quality of micro-array scans in a FE batch against a given metric set • Create a Batch summary chart from each FE batch for a given metric set • Create a QC Chart of extractions from an FE batch for a given metric set

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Installation

The first step in using the QC Chart tool is setting up the central repository or a database. The QC Chart tool operates by storing all the data in this repository so all the menus except the ‘ConfigureÆDatabase Settings’ menu are disabled as shown below.

The database is configured as follows: 1. Click on ‘ConfigureÆDatabase Settings’ menu or just press Ctrl + D. 2. The following dialog is launched



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3. A ‘QC Chart Database Settings’ dialog is launched. 4. In the ‘SQL Server Instance Name’ edit-box type in the machine name along with the database that FE uses. E.g.: ps0546\AGTFEDB where ps0546 is the machine name. For the users it will be \AGTFEDB. NOTE: To find out the machine name; Launch ‘StartÆRun’ and type in “cmd”. At the command prompt type ‘hostname’. Alternatively, one can find the SQL server name in FE by choosing the menu: Help>About:

5. Copy the name after “SERVER=” and before “;DATABASE” and past into the “SQL Server Instance Name” field of the QC Chart configuration UI. 6. If the tool is launched for the first time, choose the ‘Create new database’ radio button. In the ‘Specify folder name to store database files’ hit the Browse button and choose the default database path. 7. In the User Info frame, type ‘sa’ in the user-name edit box and ‘welcome’ in the password edit box. 8. If the user needs to map the tool to some other user’s database, re-launch the DB configuration dialog by pressing Ctrl+D or choosing ‘ConfigureÆDatabase Settings’ and change the field in the ‘SQL Server Instance Name’ edit box to the appropriate machine and hit the ‘Done’ button. Make sure that the ‘Map to existing Database’ radio button is selected. 9. Hit the ‘Done’ button to save the configuration. If the database is properly set all the relevant menus will be enabled in the tool.

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Loading Data



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The QC Chart tool needs extractions imported in its database to create meaningful metrics and thresholds. Loading the tool with extractions consists of extracting the Stats and FEParameter data from the output text files of FE. Importing the FE output text files can be done either by selecting the files individually or by specifying the base folder for recursively importing all the FE text files within all the sub-folders. To import the FE text files by individual selection, choose ‘FileÆImport FilesÆFE Files…’ or press ‘Ctrl + F’.

The ‘Browse FE files’ dialog will be launched which will allow the user to select FE text files individually as shown below. Hit the ‘Open’ button to import the extractiondata from the selected files.

To import files recursively specify the base folder by choosing ‘FileÆImport Files Recursively’ from the menu or by pressing ‘Ctrl + R’.

The following ‘Browse for Folder’ dialog will be displayed as shown below.



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Choose ‘OK’ to start importing. If there are a large number of files in the sub-folders this operation may take some time. It takes about 15-20 minutes to import 2000 FE files. Sometimes the files that are chosen may not be FE output text files. In such cases, the following dialog displays all the files that the tool classified as invalid in a list box as shown below.

Choose “OK” and the import will continue. The import process creates a “row” or “record” for each unique extraction. If a barcode has been extracted with various FE protocols, these will result in unique



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records, as the extraction name is created from both the barcode and the name of the protocol.

Two types of data are imported: - All Stats fields from the extraction - Selected FEParameters fields are also imported to aide the user in defining queries of the extractions (see Chapter 15 for a list of imported parameter fields).

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Associate Annotations

Users can create annotations for the extractions that exist in the QC Tool repository and import the annotations with the tool. Purpose: annotation associated with an extraction can be used to filter for that extraction in queries, discussed below. The annotation can also be used to organize data in QC charts produced by the QC Chart Tool; for instance, to sort extractions, or to indicate different subsets of extractions by color-coding or by shape-coding. The user can add annotations by one of three different methods, 1) Exporting all database information, editing and re-importing 2) Exporting a subset of the database information, editing and re-importing 3) Generating a new tab-delimited text file containing the annotations Each method is described below

Export-All Method 1. Once extractions are loaded into the QC Tool repository, all barcodes (and their associated FEParams and Stats) can be exported in the QC tool by choosing ‘FileÆExport Database’ or Ctrl + A. This method will result in the export of every extraction in the repository. 2. Custom annotations can be entered by opening the text file in a program such as Microsoft Excel. Add additional custom columns (with descriptive column header names in the first row) and array annotations within those columns as desired. Column header names must not contain any space characters. It is imperative that the ‘BarCode’ column is left exactly as exported, or the reimportation into the tool will not work. See the example below:



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BarCode 251209751852 251209751871 251209751873 251209751882 251209751884 251209751886 251269421651 251269421653 251269421655 251269421658 251209751887 251209751890

Project STG_1 STG_1 STG_1 STG_1 STG_1 STG_1 STG_1 STG_1 STG_1 STG_1 CGH_prep CGH_prep

Experiment GE1_Human_June06 GE1_Human_June06 GE1_Human_June06 GE1_Human_June06 GE1_Human_June06 GE1_Human_June06 GE2_Mouse_May06 GE2_Mouse_May06 GE2_Mouse_May06 GE2_Mouse_May06 Compare 2 preps Compare 2 preps

RedSample h1 h1 h1 h2 h2 h2 m1 m1 m2 m2 k1 k1

GreenSample h2 h2 h2 h1 h1 h1 m2 m2 m1 m1 k2 k2

Polarity 1 1 1 -1 -1 -1 1 1 -1 -1 1 1

Comments

re-scanned

trainee trainee

3. Delete all columns (FEParameters and Stats) other than “BarCode” and useradded annotations before re-import into the tool. 4. Confirm that Excel has not changed the formatting of the BarCode column. If the formatting has changed, change it back as described below. The BarCode format needs to be modified to allow correct re-import into the QC Tool. To do this, highlight the BarCode column,

then choose the Excel menu item: Format>Cells>Number and choose Category = Number and choose “Decimal places” = 0, as in the following screen shot.



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Stringency v4 v4 v4 v4 v4 v4 Legacy Legacy Legacy Legacy v3 v3

Now, the BarCodes will be in the correct format for re-import into the QC Chart tool.

5. After creating the annotations save the file in tab-delimited-text (TDT) format. Excel may prompt with the following screen:



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6. Choose “Yes” 7. Once the annotations are created and saved in TDT format, they can be imported into the tool by choosing ‘FileÆImport FilesÆAnnotated Extractions’ or Ctrl + E.

Export-Subset Method A QC repository may consist of extractions that already have been annotated, as described above. If new extractions are added, the user can create a query to identify the subset of barcodes which have no annotation, and then just export those for annotation. This method will be shown in greater detail below in Section 6 “Defining Queries”, Example 3. Note: as discussed in the Export-All method, all Stats and FEParameter columns need to be deleted before re-importation into the QC Chart Tool, and the BarCode field needs to be correctly formatted. The annotation file should only have the BarCode and any user-added annotation.

Generate-New Method If there are only a few barcodes to be annotated, then the user can open a blank Excel worksheet and type in the BarCode numbers exactly as they appear in the QC repository. Proceed as in steps 2, 5, 6, and 7 from the Export-All method above.

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Defining Queries

The QC Chart tool supports a Query Builder that allows the user to select a subset of the extractions among the ones available in the QC Chart tool repository. Purpose: A query is used to define a subset of extractions that define a representative data set for use in metric and threshold development. The representative data set should optimally contain data only from similar biological samples processed under identical conditions. Use of metrics and thresholds are discussed in Section 7, “Defining Metric Sets and Thresholds”. A subset of extractions may be defined by specific FEParameter fields, or by user-added annotation fields. To define queries launch the query builder by choosing ‘ViewÆExtraction Query Builder’, by pressing Ctrl + Q, or by right-clicking in the “Extractions” pane and choosing “Add New”.



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Example 1: To choose all 1-Color extractions choose the ‘Color Mode’ from the “Column name” drop down list. In the “Operator” drop-down list choose “=” and then in the right-most box, choose ‘1-Color’. Then hit ‘Add’. The SQL for that query will be displayed (e.g. “ColorMode = 0”. Click the ‘Verify’ button to ensure that the query is valid. If the query is valid the ‘Show Results and Save’ buttons will be enabled. Clicking the ‘Show Results’ button will display the results for the current query. The user can save the query with any user-defined name. The following screenshot shows this first example.

Example 2: The user can create composite SQL queries by combining then query clause with ‘AND’, ‘OR’ and ‘NOT’ operators. The query clauses can be grouped together by adding braces. The braces buttons are enabled after the user clicks on a query clause in the list box. An example of choosing all 2-color extractions that are also GeneExpression (e.g. Filtering out 1-color and CGH arrays) is showed below: Choose 2-color extractions:



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Choose “AND” button then choose Gene expression extractions:

Choose “Verify”:



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Now, the user can choose “Show Results” to see the returned set of extractions. If this is a query that should be saved, then enter desired name (e.g. “GE2”) and choose “Save”:



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Example 3: As discussed above, one may wish to find all barcodes that have not yet been annotated. One can do this by filtering on a user-added field, such as “Project” and finding records where this field is null as shown below.

Choose “Add”, “Verify” and enter name to save:



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The query now shows in the extraction pane. Selecting it will display all records that have no “Project” annotations, as shown below.

Once the results of the Null_Project are displayed (e.g. 8 records in above example), these results can be exported by right-clicking the name of the query and choosing “Export results to file…”. Now, the file can be opened and annotated as described in Section 5 “Associate Annotations”, “Method 1).

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Defining Metric Sets and Thresholds

Purpose: Metrics are defined in order for the user to follow desired statistical values across a set of extractions. These metrics can be associated in a metric set. In addition, the user can use this tool to specify thresholds for each of the metrics. This ‘Configure Metrics and Thresholds’ dialog implements the concepts of defining a metric set and thresholds. This dialog is accessible through ‘ConfigureÆMetrics’ menu or pressing ‘Ctrl + M’.

The ‘Configure Metrics and Thresholds’ dialog is shown below…



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7.1

Defining Metric Sets

A metric-set is a collection of one or more metrics and an associated query common to all the metrics. To add a metric set, right click on the white space below the ‘Metric Sets’ label. Then choose ‘Add Set’. A new pop-up appears asking the metric-set name. Choose any suitable alphanumeric name and hit OK.

7.2

Defining Metrics

A new metric can be defined by choosing ‘Add New…’ from the Metric drop-down list in the ‘Configure Metrics and Thresholds’ dialog. The following ‘Add Metric’ dialog is launched …



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The drop down list shows all available metrics from the FE Stats table output. The user can select one of these calculations, or may define a new metric as an expression. A metric expression can be created using the metric columns and operators as shown above. For example, FE calculates a slope for the eQC spike-ins (observed vs. expected Log Ratio). Depending upon the hybridization, this spike-in mixture may be present as “+1” or “-1” polarity. If it is “-1”, then any threshold that is set (e.g. Slope > 0.85), will not pass. Thus the user needs to make a derivative metric by taking the absolute of the slope. To do this, first choose the “Abs” function in the dialog. This yields “Abs( “ in the “Metric Calculation” pane, as shown below:



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Now, from the drop down list, choose the statistic “eQCObsVsExpLRSlope”, choose “Add”, then choose “ )” to finish the expression, as seen below:



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Once the metric expression is created, it needs to be validated: choose “Validate” button. After the expression passes the validation, the “Save” button is enabled. Choose a name to save, as shown below:



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The saved metric will now appear in the ‘Metric drop-down’ in the ‘Configure Metrics and Thresholds’ dialog.



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To associate a metric with a metric set, first choose the desired metric from the drop down list. Next, a query needs to be associated with the metric set. This query will filter for the appropriate extractions from your database, so that just the data from the queried extractions will be used in calculation of various statistical summary values that may be used in setting thresholds, described in the next section. For this example, the “Extraction Query” chosen from the drop down list is “GE2”, as the metric set will be used with two-color Gene Expression extractions:

Now, the metric needs to be associated with the desired metric set. Highlight the appropriate metric set, e.g. “Test_1”, then, then choose “ QC Metric Set Browser. Right-click in the pane and choose “Import” and browse to the desired metric set. After import, double-click on the metric set to see the metrics, thresholds and any mandatory metrics that are defined by this metric set.



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Once a metric set has been imported into FE, it is available to be associated with any FE project. To associate a metric set with a project, choose the desired metric set from the drop down box “QC Metric Set” in the “Other” section of the FE “Project Properties” tab:

When this project is run, the metric set will be used to yield the following output: 1. QC Chart: a run-chart type view showing extractions statistics for metrics: a. Chart visible on screen showing all extractions on X-axis with value of metric on Y-axis. If there are multiple metrics in the metric set, the plots will be stacked. b. The QC Chart can be printed or saved as text file c. The key showing where barcodes are plotted along the X-axis can also be printed 2. Project Run Summary: new information a. If thresholds are associated with the metric set, will show how many metrics were in range for each extraction b. If an evaluation metric was associated with the metric set, will show how if the extraction is in range, or needs evaluation by the user 3. QC Report: new information a. Header: If thresholds are associated with the metric set, will show how many metrics were in range for each extraction b. At the bottom of the QC Report, will show a table listing each metric and the value calculated for that extraction



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c. If thresholds are associated with the metric set, will show: i. Value of threshold ii. Color-code which metrics were in range

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Overview of Feature Extraction & QC Chart Tool Connection

FE 9.1 Metrics and thresholds appear in FE 9.1 QC reports

QC reports

Tab text data

Performance data: median signal intensities, # saturated features, S/N

Run Charting DB Define Metrics and Thresholds

Query and Presentation Capabilities



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Feature Extraction FEParameter fields used in QC Chart Tool

BarCode ExtractionName UserName ExtractionTime ComputerName Version ScanFileName PatternName DesignFileName Protocol_Name Protocol_Date Scan_ScannerName Scan_Date QCReportType ColorMode Scan_NumChannels Scan_MicronsPerPixelX Scan_MicronsPerPixelY Grid_Name Grid_Date Grid_NumRows Grid_NumCols Grid_NomSpotWidth Grid_NomSpotHeight



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QC Chart Tool guide - Agilent Technologies

QC Chart Tool guide - Agilent Technologies

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