Download the Sequencher Demo today!

Release Notes

Version 4.6 for Windows

Sequencher’s development has been and always will be a partnership between our users and Gene Codes. In appreciation for your communication, we present Sequencher 4.6.

What’s New

• Variance Table – A powerful tool for SNP analysis that displays the comparison of a group of sequences or consensus sequences to a specified reference in a table. Each cell of the Variance Table is linked to its original data so that you may edit and validate each putative difference. You can also export the Variance Table contents in a tab-delimited format.
• Features - Sequencher now imports GenBank features! You may designate a key for each feature that describes what type of feature it is, and define how to display each type of feature you import or create by using the new Define Feature Key Default Styles Feature, Motif User Preference. For instance, you can assign styles to distinguish introns from exons and variants from sequencing errors.
• Confidence – Sequencher now displays confidence and summary confidence information in the Project window, the Sequence Editor, and the Sequence Get Info window, so you can better monitor the quality of your data.

What’s Improved

• Translation – Sequencher now translates aligned sequences more accurately by ignoring placeholder gaps. AT:G now translates as Methionine rather than as an unknown amino acid.
• Five Enhanced Functions – We added power and flexibility to four popular Sequencher functions: The contig Summary view has a Compare To: Reference option. Sequence > Split After Selection splits every fragment in a contig. Contig > Create New Seq From Consensus has more gap options. Motif Definitions are now remembered between each launch of Sequencher so that you only have to set them once. We have also enhanced the view and sorting capabilities of the Assemble Interactively window.
• Customization – Since there is so much diversity in how scientists use Sequencher, we have given you greater control of your working environment to customize the workflow to fit your needs. This release lets you define the default positions of your windows and remembers settings for format and consensus options.
• Ease of Use – The items that you use frequently are more accessible, and we have made Sequencher more responsive to standard keyboard commands.
• Minor Enhancements and Fixes – In response to your feedback, we’ve made many small improvements. Sequencher works with more import and export formats. We strengthened the printing of the restriction map, and we responded to the feedback that you gave us in response to 4.5.

What’s New

Variance Table

In Sequencher, you have long been able to compare two sequences in the same contig to generate a list of differences. Many have depended on the export of these differences for tracking differences in a database. In the Variance Table in Sequencher 4.6, we have expanded both the display and export of differences in a contig or contigs.

Batch Sequence Comparison

There are several Compare To... commands under the Sequence menu - Consensus, Reference Sequence, and Top Sequence. Each command makes the appropriate comparison to your selected sequence. In Sequencher 4.6, the Compare To command still functions as it did in 4.5, but now it works with multiple selections to create a single table of differences. You can select more than one sequence from within a contig, or you can just select the entire contig from the Project window.

Selecting any of the Sequence > Compare To… commands will generate the Variance Table.

There are three sort buttons in each Variance Table. The two Total sort buttons sort the columns or rows by the total number of differences in each. They sort in ascending and descending order. You can also sort all rows by position, in both ascending (5'- 3') and descending (3'-5') order by clicking on the button above the position numbers. In the example above, the text on this button is “Reference”, but this text will change depending on the comparison command used to generate the Variance Table. The button in the lower left corner of the Variance Table expands and collapses column widths to optimize the display of the table. Click on this button - - to toggle through three different column width options, single base, full name, and partial name.

The arrow keys allow you to navigate in the Variance Table. The simple arrow keys advance you from one cell to the next. If you want your selection to focus on differences, to skip over gaps, use the arrow key with the Alt key. When you navigate in the Variance Table, a beep will sound when you arrow beyond the edge of the table.

The comparison range displayed at the top of the Variance Table, [ 5..756 ] in the above example, describes the bases in the contig involved in the comparison. When reporting differences in a table, it is important to distinguish between sequences that have no differences at a given position, blank cells in the Variance Table, and sequences that have no data at a given position. In the Variance Table, Sequencher distinguishes sequences that do not extend the full length of the comparison range by adding a pink background to the header and footer of the sequence column, as can be seen in sequence Four in the Version 4.6 Variance Table on the previous page. A pink X fills the cell at the base positions where there is no data coverage. Note: it is possible for a sequence to have a partial coverage, a pink header and footer, but not have any pink X’s in the column. This will happen whenever a sequence has partial coverage, but there are no differences reported for the other sequences at positions outside of the covered range.

Under the View menu, you have a variety of display options: Base Edits As, Display Color Bases, Display Features, and Display Base Confidences. These options affect the display of the Variance Table just as they affect the display of your data in your Contig and Fragment Editors.

In addition to expanding the Compare To… command for sequences, we have also added a new command for comparing multiple consensus sequences to a common Reference Sequence. It is a powerful tool for SNP analysis and the natural second step after you assemble To Reference by Name. To build the table, select one or more contigs in the Project window that have the same Reference Sequence. Then, from the Contig menu, execute the new Compare Consensus to Reference command. As with the sequence based Variance Table, pink X’s and pink shading in the header and footer cells will indicate when the consensus sequence does not cover the span of the Reference Sequence. Confidence values and features exist only in fragments, not in the consensus sequences, so the View > Display options have a more limited role in the display of Variance Tables based on multiple contigs.

Review Mode

The Review mode of the Variance Table connects the data in the Variance Table with the data in the underlying sequences or contigs. When you click on the Review button in the Variance Table button bar, or when you double-click on one of the cells in the Variance Table, Sequencher opens the associated Contig Editor window and Chromatogram window. The default layout arranges the 3 windows so that you can view all 3 at the same time.

When in Variance Table Review mode, as you navigate through variances using either your mouse or your arrow keys, Sequencher updates the display in the editor windows to match the selection in the Variance Table.

The Variance Table provides a snapshot of the differences in your selected sequences or contigs. While you can edit your data with the Variance Table open, the table will not reflect your edits until you click on the Refresh button. The Refresh button on the Variance Table allows you to recreate the existing table without having to go back to the Project window and re-execute the original compare command.

The Export button at the top of the Variance Table window allows you to use the contents of the Variance Table in other programs. You have two available export formats, Tab Delimited Table and Individual Reports. The Tab Delimited Table imports easily into a spreadsheet or database, and the data include the comparison coverage range for each sequence. The Individual Reports are in the same format as the export of the comparison report from previous versions of Sequencher. Therefore, you will still be able to use the Variance Table function even if you have a data pipeline that depends on our previous export.

You can choose to export the contents of the entire Variance Table or you can select specific rows or columns.

Options

The Options button allows you to modify the differences that are displayed in your Variance Table. One option insures that you display the positions that contain ambiguous bases in the Primary sequence, even if all of the sequences in that row match the ambiguous base. In the figure below on the left, you’ll note that the total number of differences for these rows is zero. The second option lets you exclude the differences that occur over the range of a large gap in your Primary sequence. In the picture on the right, the Variance Table excludes the differences between 53.1 to 53.10.

Features

In Sequencher 4.6, when you import a sequence in the GenBank format, you will automatically import the features, including the range and the feature keys. Some of the feature ranges described in GenBank are ambiguous, and therefore, not easy for Sequencher to interpret. Upon import of features with these ambiguous ranges, Sequencher will list any features that it cannot import in a printable dialog.

In Sequencher, you have always had the ability to assign a feature to a sequence selection. The legacy properties of our features include the range of bases, the feature name and the feature style. Sequencher 4.6 provides a variety of tools to help you better manage your features. Primarily, you will now be able to assign a Feature Key in addition to the standard feature properties. Feature Keys are attributes of the features associated with sequences published in GenBank, EMBL, and DDBJ. A drop down list of the standard Feature Keys is supplied in the two menus where you assign features in Sequencher, Mark Selection as Feature and Edit Features. In addition to the menus where features are assigned, the Feature Listing also displays Feature Keys.

One advantage of Feature Keys is that they can help you manage the display style of features that share a common key. In the Feature, Motif User Preference pane, you can assign a default style for the display of features in a feature key-specific way. For instance, the default display for the codon feature key is underlined, blue font, and with the display of the protein translation.

Confidence

Since version 4.1, when Sequencher started importing confidence scores with base calls and chromatograms, each release of Sequencher has furthered the use of confidence scores in sequence analysis. Release 4.6 is no exception. You will first notice that the Project window list view has a new column, Quality, that displays the percent quality of each sequence that has confidence scores. The Quality value is the percent of bases in a sequence that are above the low quality threshold as set in the Confidence User Preference pane. The default setting is 20. As with the other headers in the list view of the Project window, clicking on the header will sort the data in the Project by that attribute.

The Get Info window for a fragment now includes a base count for each of the three confidence ranges.

When bases that have confidence scores are selected in the Sequence Editor, the window header area of the editor displays the number and percentage of Low Confidence bases in the selection in addition to the % ambiguous information that Sequencher has always displayed.

In the above sequence, ten bases are selected, and one is both ambiguous and low confidence, as noted in the text above the sequence.

What’s Improved

Translation

In Sequencher 4.6, we have modified the way Sequencher interprets the gap character when translating nucleic acids to amino acids. Sequencher will now ignore gaps when translating sequences.

Translation in Sequencher 4.6:

Translation in Sequencher 4.5:

In the Sequencher 4.6 translation, Sequencher now correctly ignores the gap and translates the codon GC:C as R. Note that the position of the Amino Acid has shifted from the first base in the triplet to the second. In contrast, the Sequencher 4.5 translation translates GC: as a ? and then shifts the frame of the translation.

Five Enhanced Functions

Summary

The Summary in Sequencher lets you view the contents of your contig in a printable and exportable format. In 4.5, the Options only let you illuminate the differences in the fragments relative to your consensus sequence, but in 4.6 you can compare your sequences to your Reference instead. The new Compare To: option lets you choose to compare the assembled fragments to the Consensus or to the assembled Reference.

Note how the Reference Sequence, the one with the “R” in the icon, is now displayed below the dotted lines, and that the Matching Bases As Dashes option displays all the bases that differ from the Reference. The Compare To: Reference option also works when you are comparing the translation of the aligned sequences.

Split After Selection

The Split After Selection command on the Sequence menu lets you turn one sequence fragment into two, one 5’ and one 3’ of the selection in your sequence. Prior to Sequencher 4.6, this command functioned only in the fragments and thus needed to be repeated if multiple sequences needed to be split at the same position. In Sequencher 4.6, you can now apply the Split After Selection command to the entire contig by simply making your selection in the consensus sequence.

Create New Sequence From Consensus

The Create New Sequence From Consensus dialog has always given you the option to remove gaps when creating a new sequence from a consensus. However, there are two different kinds of gaps: small and large. Small gaps in a consensus sequence indicate a location in the contig where a minority of fragments carry an insertion; these are rarely useful in a consensus sequence. In contrast, there are a variety of reasons why a consensus might contain large gaps of ten or more; and, for some users, these gaps provide useful information. In previous versions of Sequencher, you could not eliminate the small gaps without also eliminating the large gaps. There is now a new option called Retain Large Gaps on the Create New Sequence From Consensus dialog that allows you to retain or remove large gaps independently from insertions. The new option is set to remove all gaps by default, but Sequencher will remember your preference if you change the settings.

Motif Definitions

The Motif Definitions… command under the Window menu will bring up the Motifs dialog where you may define strings of bases that Sequencher will then highlight in a display style that you also define. In Sequencher 4.6, we enhanced the Motif function by increasing the number of bases that you can define as a motif, from 25 to 50, and by persisting your motif definitions in future Sequencher sessions. We also insured that the motif files that you create and save will be compatible across platforms and that Sequencher 4.6 can support files created with earlier versions of Sequencher.

Assemble Interactively

We have expanded the Assemble Interactively window, so you can view your data more easily, and the contents of the window now sort, so that the most closely related sequences, those with the highest %match, are at the top of the list.

Customization

Resizable Columns in the List View of the Project Window

When you choose the option to View > Project Icons As > A List, you can now customize the size and presence of the columns in your project window. 4.6 features two new columns, %Quality, which is on by default, and Sample Name, which isn’t. The new resizable column headers insure that you can read your full sequence names, even when they are longer than thirty-two characters. The column that sorts the list is highlighted.

Remember Window Layout

When working in Sequencher, it is critical to optimize the positions of your windows to take greatest advantage of your screen real estate. Sequencher has always had default window positions to facilitate working in the most standard types of projects, but we recognize that you may prefer to set these defaults to better suit your projects. To this end, you have two new menu commands in Sequencher 4.6 under the Window menu, Remember Window Layout > Contig Chromatogram and Remember Window Layout > Variance Review. Each lets you define the default positions for the Contig Editor and Contig Chromatogram windows and the Variance Review command also sets the default window position for the Variance Table.

To take advantage of Remember Window Layout, first organize your windows and then execute the appropriate command. Thereafter, every time you select the Show Chromatograms command button or the Variance Table Review command button, Sequencher will open windows in your layout positions. An example of the default window arrangement and a possible alternative layout follow.

The vertical arrangement of windows on the right provides a display conducive to viewing contigs with deep coverage.

Remember How I Like to Work - Ruler Display and Scrolling

Sequencher 4.6 also helps you to control your workspace when you are analyzing unassembled fragments. Sequencher remembers the last used individual chromatogram window size and whether you prefer horizontal or vertical scrolling. Sequencher 4.6 also remembers if you want to display your Ruler. If you leave the Ruler on in the Sequence Editor or the Contig Summary, Sequencher will continue to open these windows with the Ruler on.

The Consensus Calculation method persists with your project in Sequencher 4.6. When you open a project, Sequencher’s consensus calculation is set to that of the opened project - Inclusively, By Plurality or to Forensic Standards.

Ease of Use

Open Recent File command

Our computers have so much storage capacity that we can spend lots of time looking for a project that we were just working with. To help you work more efficiently, our new File > Open Recent menu gives you a list of up to 4 of your most recently accessed projects.

AbN = Assemble by Name

The Assemble by Name function, introduced last year in Sequencher 4.5, has quickly become part of the standard operating procedure in Sequencher. The most common request we received from new users was to “please make the function more accessible”, so we have. There is a new button for Assemble by Name, AbN, on the Project window button bar. You still change the Assemble by Name Handle selection from the Assembly Parameters window, but you can turn Assemble by Name off or on directly from the Project window. Now you can use Assemble by Name to make 50 contigs from 50 clones in the click of a button, and then, without accessing the Assembly Parameters, turn off AbN to assemble the consensus sequences of the contigs with Assemble Automatically.

Shift+Control Click Selections, the Scroll Wheel, and More…

Since Sequencher was first launched, Gene Codes has added many new functions to facilitate DNA sequence analysis. We also use upgrades as an opportunity to harness a number of standard keyboard and mouse tools to facilitate your work. In Sequencher 4.6, you can select a range of fragments in a contig or in a Project window by using Shift+Click. You can still make discontiguous selections using the Control+Click. You will also find that if you have a Scroll Wheel on your mouse, this will now function in Sequencher 4.6.

Arrow keys now navigate your selection in the Project window, and you can use the Delete key or the Backspace key to remove items from your project. You will get the same alert that you now do when you move the items to the trash or execute the Edit > Remove From Project… command.

Eliminated Dialog in Show Chromatogram Window

Previously, you produced an error message in the contig chromatogram window when you executed the Show Chromatograms command in a region without trace data. That error message, below on the left, is replaced in Sequencher 4.6 with a more informative Contig Chromatogram window, below on the right.

Better Default Settings

We introduced the Realigner and Prefer 3’ Gap placement options in Assembly Parameters in Sequencher version 4.2. Initially, we set the default values for these parameters to “off”. However, the feedback that we have received has been so positive that, in Sequencher 4.6, we’ve changed the default settings to “on”.

Minor Enhancements and Fixes

Import and Export Options

• The Nexus Interleaved format for exporting contigs has always been important for those doing Systematics and Phylogenetics. In response to customer requests, we have added the Nexus Sequential format, which is more easily imported into some software.
• Previous versions of Sequencher incorrectly imported the concatenated Fasta files from the dbSNP database. This was due to unexpected base space characters in the file. In 4.6, Sequencher throws away space characters on import, and dbSNP Fasta files now import correctly.
• We have reduced the probability of creating an error when you save a project to a network volume.
• Double-clicking a project icon will open that project in a new Sequencher instance. If you already have a project open, you will be given the option to save your work. The previous response to double-clicking was to import the data, which you can still do by dragging the new project onto the open Project window.
• Sequencher no longer generates a file error when exporting a contig in the CAF file format when the number of characters in the sequence name exceeds 32. The export now succeeds and the bad side effect of reverse & complementing the top-most fragment no longer occurs. However, Sequencher is still not able to export the complete sequence name if the characters in the sequence are longer than 32 characters, but now it will truncate the name rather than generate an error.

Fixed Crashes Reported in Version 4.5

• Sequencher no longer crashes when scrolling backwards through large gaps.
• Sequencher no longer crashes after opening a contig that was assembled using mindlessly join all to right.

Falling into Demo Mode

• Many Macintosh computers use two operating systems, OSX and Classic. When both systems run simultaneously, the Classic OS monopolizes the USB port, stealing the Sequencher dongle, and thus causing Sequencher to fall into demo mode. In version 4.6, we circumvented this problem so Sequencher runs in OSX without falling into demo mode, even if Classic is running.

Cut Map

• Where reasonable, cut site maps will print on one page.
• When you print a restriction map, the header distinguishes between “Sequenced” and “Unsequenced Strand”.
• Sequencher now recognizes and displays restriction enzyme cut sites at the 3' most ends of DNA sequences.
• In addition to adding or deleting enzymes from the default enzyme list, you can now modify and save Sequencher’s default restriction enzyme selections.

Restored Correct Behavior

• In 4.5, we discovered that the Print Trace in One Page… command actually printed on two pages. In 4.6, this command yields the entire chromatogram sized to cover a single page.
• We had several reports from Window’s users who found that they had to click twice on the Show Chromatograms button in order to open the trace window. The two-click requirement, which turned out to be an artifact of working with maximized windows, has been restored to one click.
• On Windows, it is once again possible to launch multiple instances of Sequencher.
• Editing one feature in a fragment no longer affects the placement of downstream features in the same fragment. Also, Sequencher detects and corrects out of bounds feature locations.
• The Select > Next Ambiguous Base command now ignores positions where the only coverage in the contig is from the Reference, i.e. no disagreements, ambiguous bases, or low confidence scores.
• It was recently discovered that the Contig Overview map did not display start or stop codons when they occurred immediately 3’ of a gap. This was fixed in 4.6.

Other Improvements

• The Select > Next Ambiguous Base command is sensitive to disagreements, gaps, ambiguous, and low quality bases. In 4.6, low quality data positions that you have already edited will no longer be included in the selection criteria. We also increased the speed and efficiency of the selection.
• Scrolling in the overview of large contigs is faster.