Analyzing a Project without a Reference

Transcription

1 Applied Biosystems Variant Reporter Software v1.0 Analyzing a Project without a Reference Quick Reference Card This quick reference card summarizes the procedures for setting up a project in Applied Biosystems Variant Reporter Software v1.0. Use this workflow to analyze sample trace files and identify individual variants. For comprehensive information on Variant Reporter Software v1.0, see the Applied Biosystems: Variant Reporter Software Online Help and FAQs Variant Reporter Software Getting Started Guide Pre-Analysis Workflow 1 Import and assign traces into amplicons (Optional) Specify the analysis parameters 2 Analyze the project

2 Setting Up a Project without a Reference Importing Traces To import traces into Variant Reporter software: 1. In the Dashboard, click New, then click in the Project View (Task Action toolbar). 2. In the Import Traces dialog box, select the folder or file that contains the traces you want to import, then click. 3. Verify that the traces fill the Traces to Add list box in the Import Traces dialog box, then click OK. Note: You can also drag trace data directly into the Variant Reporter software. Open the source folder or file, then drag traces into the Import and Manage Traces pane in the Project View page. Assigning Traces Imported sample files have to be organized into specimens and amplicons. To assign the imported traces: 1. Click when prompted. 2. Select the appropriate delimiter (comma, period, dash) to separate the specimen name from the import string. 3. Select the appropriate delimiter to separate the amplicon name from the import string. 4. Verify that your selections appear in the Preview Trace Auto Assignment pane (below). Specifying Analysis Parameter Settings (Optional) 1. Open the Set Analysis Parameters page, then expand the section for each analysis parameter. 2. Use the default settings or change as needed: Mobility File set to Use Mobility File from Trace Mixed Base Threshold set to 25% Analyzed Data Scaling set to True Profile 3. Use the default settings or change as needed: Trim Using Quality Values Mask M13 universal sequencing primers to exclude M13 primers from the clear range Mask amplicon primers to exclude amplicon primer sequences from the clear range 4. Use the default settings or change as needed: Filter traces with a minimum Trace Score < 25 Filter traces with a median PUP Score < 4 Filter traces with % Expected TRL < 50% Note: You can change the default settings of each analysis parameter in its expanded section. Click at any time to return to using the recommended settings. Alternatively, you can load a previously saved analysis parameter file directly into an open project. Your list of saved analysis parameters is displayed in the Dashboard. Analyzing the Project To analyze a project: 1. Click. 2. Click OK when the following message is displayed: 5. Click OK. Note: If your trace-naming convention does not contain both an amplicon and a specimen name, click. Note: The software generates a reference based on the longest trimmed read length (TRL) for each amplicon in the project. The newly created reference is marked with an asterisk. Page 2

3 Post-Analysis Workflow (Optional) Review project quality 3 Review variants 4 Print/export reports Reviewing Project Quality (Optional) After a project is analyzed, you can review the trace results, edit variants, print reports, or export the project. If a Variant Reporter Software project meets all the quality thresholds (set in the Analysis Parameters), the software opens the Project Results Summary page. If a Variant Reporter Software project does not meet one or more of the quality threshold settings, the software opens the Project Quality Summary page. In this page, you can troubleshoot the area of failure on a project level. Note: To troubleshoot a failed project on a trace level, click the number of failed traces in the Project Quality Statistics pane, or open the Trace Quality Review page. Page 3

4 Reviewing Project Thresholds To view project threshold results, go to the Project Thresholds pane in the Project Quality Summary page. Reviewing the Amplicon Specimen Quality Grid To determine the quality of all specimens and amplicons in a project, go to the Amplicon Specimen Quality Grid to view values for the following metrics: All Avg. Trace Score Avg. of Median PUP Avg. CRL Avg. TRL Avg % Exp. TRL Avg Signal Strength Note: When reviewing any of the metrics, you can filter between the forward, forward and reverse, or reverse directions of a sequence. Reviewing and Editing Variants Reviewing Variants The Variant Review page lists all variants per amplicon that the software detected during analysis. To adjust the filter for false positive variant detection, change the setting of the Variant Score Threshold: 1. Click. 2. Reset the threshold by dragging the slider between False Positive and False Negative. Note: Moving the threshold changes the number of variants to review. 3. Click OK after you set the value you want. Select a variant in the Amplicon Variants table to see all specimen genotypes listed in the Specimen Variants pane. By accepting or rejecting a specimen genotype, you automatically accept or reject the associated variant. To accept or reject selected genotypes: 1. In the Specimen Variants table, click the specimen or specimens you want to accept or reject (in the GT Result column). 2. Click to accept the selected specimen genotype(s) or click to reject the selected specimen genotype(s). To accept or reject all specimen genotypes: 1. Click in the Specimen Variants table. 2. Click to accept all specimen genotypes at once, or click to reject all specimen genotypes at once. The Amplicon Variants table is automatically updated with one of the following statuses: Yes (all genotypes have been reviewed) Partial (some genotypes have been reviewed) No (no genotypes have been reviewed) The Positions of Interest (POI) Table displays all POIs per amplicon that the software detected during analysis. Filter the POI table by any of the following metrics: QV Mixed Bases Discrepancy User Edit Variant Potential Variant Note: A potential variant is a variant detected below the variant score threshold. Page 4

5 Editing Variants To edit bases: 1. In the Variant Review page, go to the Trace Segment pane. 2. Select the highlighted variant base in the consensus layer in the Snippet Visual. To delete bases: 1. Select the base or bases that you want to delete. 2. Click Delete. 3. In the Base Edit Results dialog box, click OK to verify the deletion. Saving a Project After reviewing and editing a project, save the project: 1. In the Application toolbar, click Save or Save As. 2. Enter a project name, then click OK. 3. View the newly saved project in the Dashboard (Projects). Printing and Exporting Reports 3. Using the mixed base IUB code, enter a new base letter to replace the basecalled letter. To insert bases: 1. Place the pointer at the insertion point between the two base letters on the consensus sequence, then click once. 2. Type the new base letter in the field, then click Enter. 3. Click OK in the Base Edit dialog box to verify the insertion. Printing Reports 1. Click to open the Reports dialog box. 2. To print a report: a. Select the report type that you want to print. b. Click Print Preview to see how the report will print, then click Print. Note: For the Specimen Report, you must select either all or the individual specimen of interest from the drop-down list. Exporting Reports 1. To export a report: a. Select the report you want to export. b. Select the applicable file format: project:.pdf or.txt quality:.pdf,.txt,.xls,.html 2. Click Export. Note: You can export multiple reports at once. Page 5

6 Exporting a Project To export a Variant Reporter Software project: 1. In the Application toolbar, click Export, then select Export Project. 2. Select the directory to export your project to. 3. Select.vrz as the project file type, then click Save. Reanalyzing a Project Reanalyze a project in Variant Reporter software whenever you: Update any of the analysis parameters (basecall, trim, or filter) Add or delete a reference (including adding or deleting ROIs, layers, or amplicons) Update the Amplicon Specimen Trace Grid (add, delete, move, unassign, or include/exclude individual traces) Note: Whenever reanalysis is required, the Project Status displays Reanalysis Required. Note: Reanalyzing a project reapplies your base edits and status review edits and auto-updates the variants in the Variant Review page tables. When you try to reanalyze a Variant Reporter software project that has a software-generated reference, the following message is displayed: IMPORTANT! Recalculating a new reference may result in the loss of previous edits in the project. When possible, specify a reference for a project. See Variant Reporter Software: Analyzing a Project with a Reference QRC. Copyright 2007, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Applera, Applied Biosystems, and AB (Design) are registered trademarks and Variant Reporter is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. All other trademarks are the sole property of their respective owners. 05/ Part Number Rev. B