Tour Guide for Windows and Macintosh Introduction
SEQUENCHER has been developed to work with a wide range of quencing applications. For example, SEQUENCHER can be ud to:
•Create asmblies for shotgun or EST quencing projects
•Edit contigs while viewing all relevant trace data
•Asmble multiple quences to a ur-defined Reference Sequence
•Detect and annotate polymorphisms
•Align cDNAs to their genomic quence using the Large Gap algorithm
•Discover heterozygous peaks
•Create difference reports for SNP discovery
•Display restriction maps, ORF maps, protein translations
•Automatically trim poor quality and vector quences
Macintosh and PC Support
SEQUENCHER is available for both Macintosh and PC platforms. The demo CD provides software for both environments.
Unlimited Trial
With the SEQUENCHER demo, you can u your own data and enjoy an unlimited evaluation period. The only functions not available are copying data to other applications, saving, exporting, printing, and reporting. Some sample data have been included to get you started, but once you have tried SEQUENCHER with the sample files, plea try using your own data so you can e precily what SEQUENCHER can do for you.
What You Will Learn in This Tutorial
The purpo of this tutorial is to guide you through SEQUENCHER‘s core asmbly and editing functions. Additional application-specific tutorials are included on your CD in PDF format. In this tutorial, you will
1.Install the SEQUENCHER demo
2.Create a new project
3.Import data
4.Trim quences
5.Asmble a contig
6.View contig asmbly
7.Edit asmbled chromatograms
8.Find heterozygotes
9.Work with a Reference Sequence
10.Translate quences to amino acids
11.Annotate a quence
12.Create a Variance Table and Report
13.Create a Translated Variance Table
14.What el can I do with SEQUENCHER?
Once you have mastered the techniques, you will be ready to explore SEQUENCHER ’s other powerful features.
Conventions Ud in this Guide
Menu items or keys that you are to lect are in bold. The purple text provides step-by-step instructions for running through the tour guide and the black text provides additional information. Greater than symbols define menu > submenu commands.
Before you start
Check that you have the appropriate hardware and disk space.
Additional Requirements will vary with your project.
MACINTOSH RECOMMENDED REQUIREMENTS:
•10.3.9 and higher
•512 MB RAM
•150 MB hard disk space
WINDOWS MINIMUM REQUIREMENTS:
•Windows 2000 and higher
•512 MB RAM
•150 MB hard disk space
1. Install SEQUENCHER Demo
•Inrt the demo CD into your machine.
•Double-click on the Sequencher 4.9 Demo installer icon.
The installer will create a Sequencher 4.9 Demo folder in your Applications (Mac) or Program Files > Gene Codes (Windows) folder.
2. Create a New Project
•Launch SEQUENCHER by double-clicking on the Sequencher Demo icon.
On your Macintosh, this will be in the opened Sequencher Demo folder. On a PC there is a Sequencher Demo icon on your desktop.
When SEQUENCHER is launched, a dialog will alert you that you are running in Demo Mode. After you hit the OK command button on the alert dialog,
SEQUENCHER will prent you with an empty Project window. This is where you import, manipulate, and display quence fragments and asmbled contigs.
New Project Window
3. Import Data
•To import data, lect Import > Folder from the File menu.
•Brow to the Applications (Mac) or Program Files > Gene Codes (Windows) then Sequencher 4.9 Demo > Sample Data > Demo Sample Data > Demo
Sequences folder.
•Click on the Choo (Mac) or OK (Windows) button at the bottom window.
•When prompted to import the 9 files, lect the Import All Files in Folder command button. The Project now contains the 9 quences.
Imported Sequences
The imported files, with their associated quality scores, are just one example of the wide variety of file types SEQUENCHER accepts for import. Note that the Quality column displays the % quality for each of the imported quences—the percent of bas that are above the low quality threshold as t in the Confidence Ur Preference pane.
4. Trim Sequences
SEQUENCHER has tools that allow you to trim imported quences bad on veral different criteria: ambiguous data, data that have low confidence scores, or data contaminated with vector quence. The trimmed data are fully recoverable within the SEQUENCHER project. To trim the low confidence quence:
•From the menu bar, choo Select > Select All to highlight all quences if they aren’t already highlighted.
•From the menu bar, choo Sequence > SEQUENCHER displays the Ends Trimming window for the default trimming parameters.
SEQUENCHER recognizes poor quality quence bad on a number of criteria. The confidence score, provided in the samples, is one of the most nsitive. Increasing the stringency of the trim criteria further increas the quality of your data.
•Select the Change Trim Criteria button.
•Uncheck all but the three criteria checked below, and adjust the values of the two confidence trims to match.
•Click OK to return to the overview for Ends Trimming.
The Ends Trimming window displays how much poor quality data will be trimmed bad on the defined criteria. You have the additional option to individually delect a fragment for trimming on either the 5' or 3' end by removing the "X" from the appropriate box below the trim graphic.
Ends Trimming
•Click the Trim Checked Items button at the top of the window, and then click the Trim button when asked for confirmation.
The data that you have removed are completely recoverable. SEQUENCHER always stores two copies of every imported Sequence, the original quence and the data as you have edited it in SEQUENCHER.
•Clo the Ends Trimming window and return to the Project window by clicking on the clo control in the upper corner of the window.
5. Asmble a Contig
Several alignment algorithms are provided with SEQUENCHER to accommodate the wide variety of asmbly applications. For this example, you will u the Dirty Data algorithm becau it is best suited for data that may include the occasional ambiguities or miss-calls generated by automated quencers. The Asmbly Strategies and the Asmble by Name tutorials in the Sequencher 4.9 Demo\Tutorials folder explain the other asmbly options.
