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Sequencher 4.10.1 Release Notes

(These release notes are also available in PDF format. To open a PDF, you may need the free Adobe Reader.)

We invite you to explore Sequencher 4.10.1, the next stage in the evolution of DNA analysis software.

What’s New

• Translated Variance Table – The Translated Variance Table summarizes all amino acid differences in a selected set of samples. Because the table is linked to the underlying sequence data, it is ideal for validating mutations that result in changes to the amino acid translation and for checking expression vectors.
• Filter the Results of the Variance Tables – You can restrict your Variance Table to just coding sequence, or just variants, or exons, or genes, or any region specified with a feature. Similarly you can define a Translation Range, even across multiple introns, for the Translated Variance Table.
• Reports – Sequencher’s new reporting creates printable versions of your data. The Reports provide analysis tools such as clustering of like samples in the Population Report and calculating the percentage of secondary peak heights in the Variance Detail Report.
• SuperPro Key support – We replaced the purple Eve3 key with the fully supported blue or black and blue SuperPro key. This change increases the compatibility of Macintosh standalone licenses on newer Macintosh operating systems.

What’s Improved

• Improved Sequence Features – Sequencher now imports GenBank features with complex locations, such as the joined and ordered locations that are common in exon and mRNA features. Sequencher also has improved tools for the import and annotation of complemented features.
• Enhancements to the Variance Table – We have added more tools to enhance the utility of the Variance Table. From column selections you can now label, add comments to, and get info from samples. You can also selectively remove columns from the table as you work.
• Better Translation Tools – The Reference translation now updates as you edit, and a new option forces the frame of the consensus translation to correspond to the Reference. Sequence Editors will now translate joined features even when they are separated by non-coding sequence.
• Contextual Menus – You can invoke context-sensitive menus in some Sequencher windows by using the right-mouse click on Windows and right-mouse click or Ctrl+click on Mac.

What’s New

TRANSLATED VARIANCE TABLE

The Translated Variance Table provides the same type of display as the original Variance Table, but the Translated Variance Table focuses on the differences that result in a change in the translation of your sequences. Each difference is displayed as the difference in the amino acid and the three bases that code for it. Unlike the Variance Table that displays matches to the Reference as blank cells, the Translated Variance Table displays matches in light grey. This enables you to see the differences in codons that still result in matching amino acids.

The numbering for the first base of the codon as well as the numbering for each of the amino acids appears adjacent to each reference codon and amino acid in the left-most column. As with the Variance Table, the two Total headers act as buttons to sort the columns or rows by the number of variants.

To generate the Translated Variance Table, there are now new sets of commands that parallel the Variance Table commands. Under the Sequence menu, you will find a series of Compare Translation To commands, Compare Translation To -> Consensus, Compare Translation To -> Reference Sequence, and Compare Translation To -> Top Sequence. Each command creates a table from the translation differences between your chosen primary sequence and your selected sequences. Under the Contig menu, Compare Translation to Reference creates a table from the translation of the consensus sequences of your selected contigs and their common Reference.

The Translated Variance Table displays the translation of ambiguous codons as question marks. The Translated Variance Table will always display a position where the Primary sequence codon translates ambiguously, even if all of the comparison sequences match the ambiguous codon.

Review Mode

Both the Variance Table and the Translated Variance Table have a Review button that allows you to link the differences reported in the table to the data that support them. When you click on the Review button in the Translated Variance Table, as with the Variance Table, Sequencher opens the associated contig and chromatogram editors. The first base of the sample codon is selected and the effected codon of the primary and sample sequence are highlighted. Unlike the Variance Table, you cannot edit your sequences directly from a selection in the Translated Variance Table when in Review mode.

Review Mode With Sister Tables

On the Translated Variance Table window there is a Bases button. On the Variance Table window there is a Translation button. When you click on either of these buttons, you will open the sister table to the one you have open. For instance, if you have a standard Variance Table open and you click on the Translation button, Sequencher will open the Translated Variance Table for the same data that you have in the standard Variance Table. If you are in Review mode, you can define how the four windows should lay out as a preference. Just arrange the windows in the configuration that best suits your work and then select Window -> Remember Window Layout -> Double Variance Table Review.

The two tables contain different but closely related data. Not all differences in the Variance Table are on the Translated Variance Table and vice versa, so selection in the two tables are not linked, but they have many other similarities. The names of the samples and the positions are identical, except the Translated Variance Table also includes amino acid numbering. A selection in either table opens the related data in the contig editors, and a Refresh of either table refreshes the data in both.

FILTER THE RESULTS OF THE VARIANCE TABLES

The Comparison Range defines the bases included in your Variance Table. Until Sequencher 4.10.1, the sequence that you selected as your primary sequence defined your Comparison Range, so if your primary sequence was a 100kb genomic reference sequence, so was your Comparison Range. In Sequencher 4.10.1, you will have numerous options to limit your comparison range to specific bases or ranges of interest.

Feature Key or Feature Defined Comparison Range for the Variance Table

Sequencher uses Features or Feature Keys in your sequence to define the comparison range of your Variance Table. Feature Keys, such as exons, CDS, and mutations, are properties of sequence features. They are either imported with GenBank features or assigned to a feature as you create them in Sequencher. A new button on the Variance Table, Comparison Range, opens the Comparison Range dialog. In this dialog, you can select the option to filter your Comparison Range and you can define the nature of the filter.

The Feature Key pull down menu lists the feature keys of the features in your primary sequence.

Your Feature Key selection then provides you with a list of features that share the same Feature Key. You can create a comparison range based on one or multiple feature selections. You can also expand the comparison range beyond the defined location of a feature to include flanking bases on both sides of your feature. This is particularly useful if the focus of your analysis is exons and the adjacent bases that may effect splice variation.

The Variance Table below has an unfiltered comparison range, "Base Positions: 1..32375".

The table below represents the comparison range of the same Variance filtered by all of the CDS features. The reported differences are restricted to those that lie within the 13 CDS features. The "Base Positions:" field in the header lists the 13 ranges of the features. The resulting table decreases the overall number of reported variants from 63 to 25. Also note that the background color of all of the differences is now blue. This confirms that each of the reported variants is at the location of a CDS feature with a blue display property.

Note: Choosing a Comparison Range from the Comparison Range dialog limits the display of the Variance Table and its export. Selecting specific rows for export is another way to limit the Comparison Range export. When you do this, Sequencher only exports the data from the selected rows, and the base positions that define the Comparison Range are described as "Selected Rows."

Feature defined Translation Range for the Translated Variance Table

As the Comparison Range filters the displayed data in the Variance Table, so does the Translation Range for the Translated Variance Table, but its function is even more significant. The Translation Range defines the reading frame of the exemplar sequences. Because it is crucial to avoid overlapping reading frames, Sequencher uses individual features, rather than the feature keys, to define the Translation Range.

A Translation Range button on the Translated Variance Table opens the dialog that allows you to define your Translation Range. This dialog requires that your primary sequence contain at least one feature. Each feature is filtered by feature key and listed by name and location. Features that are imported as GenBank joined features or features that have the same name followed by a bracketed number are listed only as one item in the Translation Range dialog. These joined features are distinguished in the list from single features, because they list the number of features in the join rather than its location. When you choose a feature from the list, Sequencher then uses the location of the feature to define the Translation Range in the Translated Variance Table. Joined features, such as CDS, are listed as one item in the selection and are translated across the features. If you hover your mouse over any of the selections, Sequencher generates a tooltip that displays the full location information of the selected feature(s). The following two examples display an unfiltered and a filtered Translated Variance Table for a data set of six samples. Prior to setting the Translation Range, there are 806 amino acid differences, some of which may be the result of frame shifts. The Comparison Range allows you to define both the frame and the specific amino acids to be translated. The filter reduces the number of amino acids in the table to 80 translated differences.

The Pink column headers indicate that the data covering the comparison range, base positions 1 to 2580 and amino acid positions 1 to 860, are incomplete. In the Translated Variance Table below, the comparison range has been defined by a misc feature, which covers bases 817 to 1036 and amino acid positions 1 to 73. When the comparison range is more narrowly defined, the data overlap the entire comparison range so the column headers are grey.

REPORTS

The Export button on the Variance Table has been upgraded to a Reports button that provides you with numerous new ways to view the data from the Variance Table outside of Sequencher. Choose one of the available reports from the list on the Variance Table Reports dialog. The report that you select will honor your selection of columns or rows. You have four reports to choose from and you can display each in either a scrollable format or in a format suitable for printing. You have four reports to choose from and you can display each in either a scrollable format or in a format suitable for printing.

To view a report, click on the Reports button from the button bar. Then, choose one of the four report options from the Variance Table Reports dialog, and click on the Open Report... button to open your chosen report in the Sequencher Report Preview.

Although, the Reports are not available in demonstration versions of Sequencher, you can see examples in the Reports section of the Help menu.

Sequencher Report Preview

Sequencher Report Preview is a new mechanism within Sequencher that lets you display and print the data in your Variance Table in a variety of report formats. You have several options within Report Preview. From the Report View window, the button bar allows you to Print, Save as PDF, change your Page Setup, or Close the report. You can also switch to the Print Preview, which will present you with a version of the report that is suitable for printing.

The Print Preview window also allows you to change the magnification of your report with Zoom In and Zoom Out options.

Variance Table Report

The Variance Table Report honors your view of the Variance Table including, color, font, and column width. One difference that you will notice is the location of the sample coverage range. It precedes the Variance Table in the editable version and follows it in the report.

Individual Variance Reports

The Individual Variance Reports option creates a separate report for each column in the table. Each separate report includes a header, which describes the conditions of the comparison, and a one column Variance Table followed by the Comparison Range Coverage details. The following example is for the first sample, N14.

Variance Detail Report

The Variance Detail Report provides all of the information available in Sequencher to support the Variance Table. Like in the Individual Variance Reports, a summary table lists the Variants for each sample or column. Additionally in the Variance Detail Report, a Detail Table follows the Variant Table for each of the described variants. The details in this table include the sequence names, orientation, and base call of any data that contributes to the calling of the Variant. If confidence or chromatogram data are available, this is also included for each sequence in the table. The chromatogram data include the specifics of secondary peak contribution and an image of the chromatogram for up to six tracelets per variant. In this example, N14 has two variants. Each is supported by two sequences with confidence and chromatogram data.

If there are deletions relative to the Reference, the confidence value for the deleted base is the average of the flanking bases.

Population Report

The Population Report is a unique summary of the data in the Variance Table. Each Population Report consists of two types of tables, one Populations table and several individual detail tables that describe each group in the Populations table.

• The Population table consolidates all columns in the Variance Table that share the same Variant.
• The row at the bottom of each column displays the total number of samples that contribute to each group.
• The column to the right of the table displays the total number of samples that have a variant at that row position.
• The detail tables report frequency for each group and describe variants and specific samples included in each group.

The following Population Report describes 48 samples of a total of 58 samples in the Variance Table. The 10 samples not included in the table were dropped because their data do not cover the entire Comparison Range.

What’s Improved

IMPROVED SEQUENCE FEATURES

In each of the last three releases of Sequencher, we have enhanced your ability to add, edit, and import features. The 4.9 release is no exception. A GenBank feature location may be defined by a simple range or a complex expression. Previous versions of Sequencher were able to import features with a simple location, but Sequencher was not able to import the more complex expressions. Sequencher 4.10.1 has eliminated almost all of these limitations. In addition to enhanced imports, Sequencher 4.10.1 has a more descriptive Feature Listing and the Edit Features dialog is also more flexible. Both of these commands are available from the Sequence menu.

Joined Features Plus

Exons and Codons, because their locations are discontinuous within a gene, are frequently described with a joined location. Sequencher imports each location in the join as a separate feature and adds a bracketed number to the name of the feature to convey its position in the join.

Note in the example extracted from the Feature Listing on the right, Sequencher’s name for the feature is "MMP9 mRNA [02]". The "[02]" identifies this mRNA as the second location in a join, which is confirmed by the Sequencher location of the feature, "From: 959 To: 1191", and the second mRNA described in the GenBank join location, "959..1191".

Sequencher uses the same paradigm for the import of GenBank features with discontinuous locations for the "order" rather than "join" modifier.

Complemented Features

Sequencher is now able to create and import features that have a complemented location. When you import a complemented feature, the Edit Features dialog and the Feature Listing window will both identify the feature as complemented. If a complemented feature has protein translation as a second strand characteristic, then this attribute will only be visible when that feature is in its forward orientation.

In the Feature Listing above, the feature location for the MMP9 gene is described twice. The first location, "From: 1 To: 7654 Complement", is the Sequencher definition and reflects the current orientation of the sequence. The second location is the location as stated in the GenBank entry and this location does not change with the orientation.

To the right is the same Feature Listing after the sequence was reversed and complemented. Note how the expression "Strand: Complement" has been dropped from the Sequencher interpretation of the location, but the GenBank location has not changed. Because Sequencher imports joined features and complemented features, it can now also import features with the modifier "complement (join(locationrange))."

Easier Access to Feature Related Windows

The Feature Listing window and Edit Features dialog are now accessible for a single sequence selection from the Project Window.

ENHANCEMENTS TO THE VARIANCE TABLE

From the time the Variance Table was introduced in 2006, we received numerous requests to expand its functionality. The following describes a number of additions to the original Variance Table functions, several of which also apply to the Translated Variance Table.

Populate All Cells Option

In response to user requests for the display of all bases in the Variance Table, whether or not they matched the primary sequence, we have added the Variance Table option, Populate All Cells. The selection of this option places a character in every cell in the Variance Table for both display within Sequencher and for export.

Access the Commands Label and Get Info from Variance Tables

The Edit->Label command allows you to mark one or many selected sequences with a name and a style. You can create a set of custom names to choose from in User Preferences, or you can choose a default name.

The File -> Get Info command provides the specifications of your selected samples and it also enables you to add information to the samples’ comments. You have always had access to these commands in your Project Window and in your Sequencher Editors. Now in 4.9, Label and Get Info are now also available for column selections in the Variance Table and the Translated Variance Table.

When you add a label to an item in either Variance Table, if the Label has a color property, then the display of name of the sequence in the Variance Table column will have that color. If you hover over the name of the sample, a tooltip provides the full name of the sample and several other attributes including the label.

Selectively Remove Columns from Variance Tables

The ability to selectively remove columns from the Variance Tables gives you more flexibility and power in the analysis of your data. If you use the Variance Table to identify clones, you can remove the clones that don’t match your Reference. If you use the table to confirm SNPs, you can label and then remove the samples as they are validated. The Remove From Table command is available from the Edit menu.

Added Display Information to the Variance Table Header

The Options button allows you to change the display of the Variance Table. The settings that you choose are now reflected in the contents of the Variance Table header.

BETTER TRANSLATION TOOLS

Reference Sequence Translation

The display of the Reference Sequence translation in the Contig Editor is now sensitive to modifications in the Reference, and will update to match the current Reference. You can toggle the Contig Editor display of the Reference Sequence translation from the View menu Reference Sequence Translation command. The 4.9 Reference translation is further distinguished from its display in 4.7 by a pale grey border that matches the lines bordering the nucleic acid Reference.

Reference Translation Toggle

You can now toggle the display of the Reference Translation from the Contig Editor. Click on the "R" icon below the consensus translation button in the Contig Editor to display or remove the translation of the Reference.

Reference Frame Translation

The consensus sequence in a contig may now be translated by 5 different commands available from either the view menu or from the translation button in the bottom right corner of the Contig Editor. The first, second, third, and all frames translations were available in previous versions of Sequencher. The new Translation view, In Reference Frame, translates the consensus so that it matches the frame of the Reference translation.

The View menu command and toggling the translation button on the Contig Editor to "r" displays the translation In Reference Frame, specifically the same frame as the Reference. This option is not available if the contig does not contain a Reference.

Translation of Joined and Ordered Features

In addition to color, changes in the font, and underlining, you have always been able to assign translation as an attribute of a Sequencher feature. The feature translation displays as a second line in the Sequence Editor. When Sequencher 4.10.1 imports joined features or ordered features, and these features are assigned a display style of Protein Translation, it translates across the discontinuous feature ranges, even when the codons are split between two locations, as in the example below.

The two joined CDS features in this sample have a display style of blue and Protein Translation. Note that the last G highlighted in the first blue feature is the first base in the GTG codon, which codes for Valine. The remaining two bases for this codon are the first TG in the beginning of the next blue highlighted region. The numbers to the left of the sequence also compensate for the discontinuous nature of the translation. The Valine at the end of the first feature is the 217th amino acid. The 218th amino acid appears 92 bases later. The (...) denotes the amino acid numbering between joined features with a protein translation.

In order for Sequencher to join the translation of two features, the names of the features must be identical followed by a number in square brackets. This is the default naming scheme for the import of joined or ordered features from GenBank, but you can also create and use this naming scheme to create joined features within Sequencher.

CONTEXTUAL MENUS

Throughout Sequencher, we have added context-sensitive menus. These are accessible with a right-mouse click on Windows and Mac or with a Ctrl+Click on the Macintosh. The following table illustrates the variety of menus accessible from different selections in a Contig Editor.

Contextual menus have also been added to the following:

MINOR ENHANCEMENTS AND FIXES

Fixed Navigation Errors

Selection now works correctly when navigating with the arrow keys in the Chromatogram Editor window. Previously, the selection skipped the base adjacent to the last edited base when arrowing to the left or right.

We eliminated conditions that sometimes resulted in the skipping of low confidence bases with the Select Next Low Confidence Base command.

Recalculation of Consensus Eliminates Error

Users reported rare instances when the Reference Sequence incorrectly contributed to a consensus sequence on export. To remove the possibility of an error, Sequencher now recalculates the consensus with each export.

Remember the Last Size of the Sequence Editor Window

In response to user requests, the size of the last open Sequence Editor window now defines the default size of the window.

Consensus Now Updates Immediately in Response to Moved Gaps

The recalculation of the consensus no longer requires a refresh of the Contig Editor window in response to the movement of gaps in the consensus sequence.