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Tour Guide for Macintosh

Introduction

SEQUENCHER has been developed to work with a wide range of sequencing applications. For example, SEQUENCHER can be used for:

Creating assemblies for shotgun or EST sequencing projects
Editing contigs while viewing all relevant trace data
Assembling multiple sequences to a user-defined Reference Sequence
Detecting and annotating polymorphisms
Aligning cDNAs to their genomic sequence using the Large Gap algorithm
Discovering heterozygous peaks
Creating difference reports for SNP discovery
Displaying restriction maps, ORF maps, protein translations
Automatic trimming for quality and for vector

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 use your own data and enjoy an unlimited evaluation period. The only functions not available are SAVE, PRINT, COPY, and EXPORT. Some sample data have been included to get you started, but once you have tried SEQUENCHER with the sample files, please try using your own data so you can see precisely what SEQUENCHER can do for you.

What You Will Learn in This Tutorial

The purpose of this tutorial is to guide you through SEQUENCHER‘s core assembly and editing functions. Additional application-specific tutorials are included on your CD in PDF format. In this tutorial, you will

Install the SEQUENCHER demo
Create a new project
Import data
Trim sequences
Assemble a contig
View contig assembly
Edit assembled chromatograms
Work with a Reference Sequence
Translate sequences to amino acids
Annotate a sequence
Create a variance table
Create a new sequence from a consensus
Create a summary report
What else can I do with SEQUENCHER?

Once you have mastered these techniques, you will be ready to explore SEQUENCHER ’s other powerful features.

Conventions Used in this Guide

Menu items or keys that you are to select 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.

MACINTOSH MINIMUM REQUIREMENTS:

9.2 and higher for Classic Mac OS
10.2 and higher for Mac OS X
128 MB RAM
40 MB hard disk space

1. Install SEQUENCHER Demo

Insert the demo CD into your Macintosh.
Double click on the Sequencher 4.6 Demo Install icon.
Enter your user password when prompted for authentication.

The installer will create a Sequencher 4.6 Demo folder in your Applications folder.

2. Create a New Project

Open the SEQUENCHER 4.6 Demo by double clicking on the SEQUENCHER 4.6 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 present you with an empty Project window. This is where you import, manipulate, and display sequence fragments and assembled contigs.

New Project Window

3. Import Data

To import sequences into this project, select Import > Folder of Sequences… from the File menu.
Browse to the Applications > Sequencher 4.6 Demo > Sample Data > T.G. Sample Data > ADARC folder.
On OS X, click on the Choose button at the bottom of the Choose a Folder window. In Classic, click on the Select “ADARC” button.
When prompted to import the 8 files, select the Import All Files in Folder command button. The 8 files contained in that folder will now appear selected in the
Project window.

Imported Sequences

These imported files, from an Applied Biosystems 3700, are just one example of the wide variety of files types SEQUENCHER accepts for import.

4. Trim Sequences

SEQUENCHER has tools that allow you to trim imported sequences based on a number of different criteria: ambiguous data, data that have low confidence scores, or data contaminated with vector sequence. The trimmed data are fully recoverable within the SEQUENCHER project. To trim the ambiguous sequence:

From the menu bar, choose Select > Select All to highlight all sequences if they aren’t already highlighted.
From the menu bar, choose Sequence > Trim Ends... SEQUENCHER displays the Ends Trimming window for the default trimming parameters.

These sample sequences are of high quality, containing very few ambiguous base calls. Increasing the stringency of the trim criteria further increases the quality of the data.

Select the Change Trim Criteria button.
Note the checked lines in the figure below and adjust the values of just these four lines.

Change Trim Criteria

The Trim Criteria dialog allows you to separately define the settings for trimming either the 3’ or 5’end based on a variety of different criteria. We trim the imported ADARC samples based on a concentration of ambiguous base calls. Had the imported data included confidence or quality scores, this would have allowed us to trim based on concentrations of low quality base calls. You will find more information on working with this type of data in the Quality Scores tutorial.

Click OK to return to the overview for Ends Trimming.

Ends Trimming

The Ends Trimming window displays how much ambiguous data will be trimmed based on the defined criteria. The user has the additional option to individually deselect a fragment for trimming on either the 5’ or 3’ end by removing the “X” from the appropriate box below the trim graphic.

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 sequence and the data as you have edited in SEQUENCHER.

Close the Ends Trimming window and return to the Project window by clicking on the close box in the upper left hand corner of the window.

5. Assemble a Contig

Several alignment algorithms are provided with SEQUENCHER to accommodate the wide variety of assembly applications. For this example, you will use the Dirty Data algorithm because it is best suited for data that may include the occasional ambiguities or miss-calls generated by automated sequencers. The Assembly Strategies and the Assemble by Name tutorials in the Sequencher 4.6 Demo folder explain the other assembly options.

Click on the Assembly Parameters button at the top of the Project window.
Accept the defaults for the Assembly Algorithm, Minimum Match Percentage, and Minimum Overlap parameters. They should be Dirty Data, 85% and 20 bases, respectively.
Optimize gap placement by selecting Use ReAligner and Prefer 3’ Gap Placement, if not already selected.
Select OK.

Once you have returned to the Project window, you are ready to begin assembly.

All of the sequence fragments should be selected. If they are not, select them now using Select > Select All.
Click on the Assemble Automatically button at the top of the Project window.
Select Close to dismiss the dialog.

6. View Contig Assembly

The Contig User Preference defines sorting criteria for new assemblies. The default preference sorts the fragments according to position, 5’ to 3’, within the contig. SEQUENCHER provides a number of alternative sorting options.

Double click on the Contig[0001] icon to open the contig Overview window.
Click on the Sort button , select the by Strand radio button, and click on OK to sort the fragments by strand.

Contig Overview

Click on the Sort button, select the by Position radio button, and click OK to return to the original sorting order.

The Overview contains three sections. The top section displays a schematic of how the fragments are assembled in this contig. The arrows indicate the direction of the fragment in relation to the assembly.

SEQUENCHER provides a Selection Marquee that allows you to navigate from within the Overview into the Bases window.

The next section provides coverage information. For instance, the consensus called around base 649 has less coverage than the surrounding consensus bases.

Below the coverage bar is the open reading frame map. Three bars marked with green flags and red lines, representing start and stop codons respectively.

Click and drag on the Selection Marquee in the Overview so that it selects the region around base 650.
Select the Bases button at the top of the window to open the Contig Editor and view the base sequences that assemble at this position.

7. Edit Assembled Chromatograms

The Contig Editor provides the tools for checking and editing sequences. It is divided into four quadrants. You can modify the appearance of the contig editor from the View menu and in your User Preferences.

Select View > Display Color Bases.
Under Window > User Preferences change Display > Contig so the Font is Monaco and the Size is 18.

Contig Editor

The two upper panels show the individual fragment names to the left with their sequences to the right. The Agent Box contains descriptive information about your sequences and your selection. The lower right panel displays consensus information including ambiguities <+> and disagreements <•>.

The Select menu provides several tools to navigate to areas of interest in the contig.

Select the base in the consensus sequence above the <•> bullet, at base position 683. The Agent box reads “3 frag bases selected at consensus position 683.”
To view the chromatograms at this position, while your selection is still on the ambiguous consensus position, select the Show Chromatograms button at the top of the window.

Assembled Trace Windows

Each chromatogram window displays the edited version of the base calls in black and above a line that separates them from the original base calls. At the left of the trace window, SEQUENCHER provides tools that allow you to manipulate how the traces are displayed. The volume control bars allow you to adjust the chromatogram peak height. The A, C, G, and T buttons allow you to turn off the display of the signal from any or all of the bases. Additional controls to format the traces are available through the Window menu under User Preferences…, Display.

The base call for ADARC4 at position 683 is incorrect. To change this “:” to “A”, with your selection still on the consensus base, type “A”. Your edit will correct the consensus and every sequence in the contig at that position.

Note that SEQUENCHER displays the edited base call in a contrasting color. You are now ready to continue editing your sequence. You can move in your contig from one region of ambiguity to the next using SEQUENCHER ’s navigational tools.

From the Select menu, choose Next Ambiguous Base, or use the keyboard shortcut, Command+N, defined next to the menu command.

SEQUENCHER jumps to the next ambiguity or disagreement in the contig. An ambiguity is defined as any base other than an A, C, G or T. Once you have chosen a selection tool, the spacebar becomes a shortcut for repeating the selection command.

Edit the consensus base and select the next ambiguous consensus position by clicking on the spacebar.
Continue selecting and editing until all the ambiguities are resolved 3’ of base 683
Use the Shift + Spacebar command to find and edit all of the ambiguous bases 5’ of base 683.
Close the Contig and Chromatogram windows by clicking on the close buttons in the upper left corners of each.

8. Work with a Reference Sequence

There are numerous potential applications for the Reference Sequence. The Reference Sequence facilitates comparative sequence alignments, defines base numbering, and boosts assembly speed. In this Tour Guide, we will use the Reference Sequence function to compare the assembled trace sequences to a known text sequence.

From the Project window, select File > Import > Sequences… and navigate to the T.G. Sample Data folder from which you imported the trace files.
Click on the file HIVSFAAA.TXT, then click on the Open button.

The new sequence, HIVSFAAA, is now in your project.

Deselect the Contig by Command+clicking on the Contig[0001] icon.
To make HIVSFAAA your Reference Sequence, while the sequence is still selected, select Sequence > Reference Sequence. The icon will now contain a capital “R” to indicate that it is a Reference Sequence.

You can use the same assembly parameters that we used for the autoseq fragments. Note that the current parameters are listed in the Project window just below the button bar.

Choose Select > Select All and click on the Assemble to Reference button.
Click Close to dismiss the Assembly Completed dialog.

The Reference Sequence is now incorporated into Contig[0001].

Double click on the Contig[0001] icon to display the contig Overview.

Immediately you can see that the Reference Sequence contributes to the contig in a different manner than the non-Reference sequences. For example, the numbering of the sequence, as it appears on the coverage bar, is negative until the Reference Sequence begins to contribute.

When the data that extends beyond the reference is not of interest, you can trim the contig sequences to the Reference Sequence.

From the Contig menu, select the Trim to Reference Sequence command.

The Contig[0001] contig now starts at base position 1. It is useful to view a Reference Sequence when editing a sample sequence. The Reference Sequence will guide you so you can critically examine the differences between the sample consensus and the Reference. Yet the Reference will not contribute to the consensus nor will the Reference be affected by edits in the consensus.

Drag the Selection Marquee in the Overview to the 5’ end of the contig.
Click on the Bases button.
Place your cursor on base position 1 in the consensus line and click on it.

Choose Select > Next Ambiguous Base.
Click on the Show Chromatograms button.

9. Translate Sequences to Amino Acids

SEQUENCHER provides a variety of ways to display the translation of DNA sequence. For instance, you can display one or all three of the reading frames below the sequence while editing.

Click on the <•> button in the lower right corner of the Contig Editor. SEQUENCHER displays the translated consensus.

The first click changes the <•> to a <1>, displaying the translation in the first reading frame. This button will toggle through each of the reading frames, concluding with the display of all three and then returning to a display of ambiguities and disagreements.

Click on the translation button until it reaches a <1> to leave the translation in the first reading frame.

SEQUENCHER also allows you to display the translation of the Reference. To view the Reference Sequence translation, do the following:

In the menu bar, select View > Reference Sequence Translation.

Contig With Protein Translation of Consensus and Reference Sequence

10. Annotate a Sequence

SEQUENCHER provides for sequence annotation by allowing you to create a feature for a single base or a range of bases. If your cursor is not on position 237, follow the instruction below to use the Select tools to move your consensus selection.

Select a base in the consensus sequence other than base 237.
From the menu bar, choose Select > Bases by Number….
In the Select Base dialog, enter 237 in both text boxes.

Click OK.

At this position, the sample sequence has a stop codon, TGA, and the Reference codes for a Cysteine, TGT.

Choose Sequence > Mark Selection As Feature.
From the drop down Feature Key menu, choose variation.
In the Feature Name box, give your feature a brief name like “T- > A, Cys- > Stop”.
The default Feature Style for a variation is red and underlined, but you may choose any style you wish.
Change the color to Cyan from the Feature Color drop down menu.
Click OK.
To view the feature annotation with the consensus sequence, click on the translation button until it is a <• > .
If the View > Display Features menu item is not checked, select it.
Turn off Display Color Bases from the View menu.

The feature will now be displayed in both the Bases view and the Overview.

11. Create a Variance Table

After you have edited the sample sequence so that you are confident that the consensus base calls are correct, you can generate a comparison report of how the sample differs from the Reference.

Close the Contig and Chromatogram editor windows.
With Contig[0001] selected, choose Contig > Compare Consensus to Reference.

Sequencher displays a table listing the differences between the consensus sequence that you edited and the reference sequence that you imported. Note that base 237 is still cyan and underlined.

You can also modify the look of this table.

Click on the symbol that looks like an open elevator button in the bottom left corner to expand the width of the columns.
From the View menu, choose Colors as Backgrounds.
From the View menu, choose Display Color Bases.

Variance Table

This report lists the differences between the consensus of one contig and the Reference, but it is also possible to create this report for hundreds of contigs, when they share the same Reference.

Double click on the cell at base position 237.

Sequencher rearranges the windows and opens the Contig and Chromatogram Editors for that base position.

Close all SEQUENCHER windows except the Project window.

12. Create a New Sequence from a Consensus

Once you have edited an assembly of sequences, you may want to perform further analysis on the consensus sequence of the contig. In the full version of SEQUENCHER, you can export the Consensus sequence using export functions in the File menu. You can also create a new sequence from the consensus within your SEQUENCHER project.

Select the Contig[0001] icon if it is not already selected.
In the menu bar, select Contig > Create New Seq From Consensus….

SEQUENCHER will provide you with the following dialog:

Click OK.

SEQUENCHER will create a new sequence from the consensus of the selected contig. Based on the checked options, the new sequence will not include gaps but will include features, and SEQUENCHER will append the date created to the name of the new sequence such as “Contig[0001] @<date > , <time > ”.

13. Create a Summary Report

The Summary Report is a printable exportable view of a contig assembly. It has a variety of formatting and display options that make it a useful means of reporting assembly information for lab notebooks, publication, or presentation.

To demonstrate the Summary Report, you will create a new contig from the consensus sequence that we just created and the Reference Sequence. This summary will contain only two sequences, but it can contain as many sequences as you can assemble (about 32,000).

From the Project window, select File > Import > Sequences…. The Import dialog will take you to the T.G. Sample Data folder.
Click on the file HIVSFAAA.TXT, then click on the Open button. You have again imported the HIVSFAAA sequence into your Project window.
Make HIVSFAAA a Reference sequence, using Sequence > Reference Sequence.
Shift+click to select both the HIVSFAAA and Contig[0001] @ <date> , <time> fragments if they are not both already selected. Be sure that you do not also select the assembly Contig[0001].
Click Assemble to Reference to assemble the selected fragments.
Click the Close button to dismiss the Assembly Completed dialog.
Double click on the new contig icon Contig[0005] to open it in the Contig Editor.
To open the Summary Report, click on the Summary button.

The default view displays the Summary Report for the nucleic acid sequence. To modify the report, perform the following operations:

Select the Options button and modify the selections to match the window below.

Click OK.
Select the Ruler button. The ruler will display below the button bar.
Set the translation to the first reading frame by clicking on the ruler’s translation button so that a 1 appears in the box.
Set the base grouping in threes by clicking the fifth ruler icon |:::|.

The resulting Summary Report gives the base position for the sequence and the Reference. SEQUENCHER also provides the translation for each of the fragment sequences and the consensus. Alternative options will display only the base or amino acid differences.

Scroll the Summary Report so you can view the bases near the feature you created.

In the full version of SEQUENCHER, you can export this report as text or print it.

Summary Report - around the position of the variant

14. What else can I do in Sequencher?

You have now tried the basic capabilities of SEQUENCHER for assembly and alignment of DNA sequences. Continue to explore the power of SEQUENCHER using your own data. We invite you to explore the additional tutorials available as PDFs on the demo CD.

Thousands of laboratories around the world have made SEQUENCHER the desktop standard for DNA assembly and alignment. Once you have worked with SEQUENCHER, you will understand why.

Contacting Gene Codes

We welcome your questions and input on SEQUENCHER. For questions regarding the use of SEQUENCHER or to purchase your own copies, please use one of the following methods to contact us:

By Email

info@genecodes.com

By Phone

In the US: 800.497.4939
Outside the US: +1.734.769.7249
Fax: +1.734.769.7074

By Mail

Gene Codes Corporation
775 Technology Drive, Suite 100A
Ann Arbor, Michigan 48108 USA