Release Notes. Version Gene Codes Corporation

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1 Version Release Notes 2010 Gene Codes Corporation Gene Codes Corporation 775 Technology Drive, Ann Arbor, MI USA (USA) (elsewhere) (fax)

2 Sequencher Release Notes What s New Option to Compute Consensus by Confidence There is a new method that can be chosen for computing the bases that make up the consensus string. Consensus by Confidence uses the base with the highest confidence score in the column to use as the consensus base. Consensus line shows confidence shading When Consensus by Confidence is used, the consensus line can show confidence shading. Create New Sequence from Consensus includes consensus confidence scores. This is only applicable if you have computed the consensus using the By Confidence method. Contig Coverage Graph A new version of the Overview shows the depth of forward and reverse coverage as a histogram. Generate a Shuffled sequence With this new command, bases of a chosen sequence are randomized to create a new sequence with the same composition but a different order. Frequency Histograms and Tables Nucleotide frequency information is now available as a report. You can generate tables or a histogram. New Import Formats Three major formats have been added to the extensive set currently available. These are FastQ, FastA aligned, and Contig Express projects. Support for Contig Express project files (the assembler from VectorNTI ) For those of you who have legacy projects assembled with ContigExpress (file extension.cep), you can import those projects into Sequencher, including sequence bases, names, contigs, electropherograms, features, confidence scores, base edits (including insertions and deletions), and comments. Consensus by Confidence in the Variance Table When the Compare Consensus to Reference command is used, you see Confidence Range colors in the cells of the table where there are variant bases. New Difference Column in the Variance Table A new column containing the total number of differences has been added to a number of the Variance Table reports. Gene Codes Corporation 2010 Sequencher Release Notes p. 2 of 13

3 Sort by Handle A new option to sort by Handle has been added to the Contig Editor Overview. New AppleScript commands Get and Retrieve project item values New AppleScript commands have been added to Get and Set labels and comments on any fragment, contig, or refrigerator in the Project Window. Comments on the Project can also be accessed through Get and Set commands in AppleScript. New AppleScript commands to access whether an item is Selected New AppleScript commands have been added to Get and Set the Selection state of an item. What s Improved Multiple Feature Listings The ability to open the Feature Listing has been extended from one sequence to a number of sequences at the same time. Chromatograms on the Variance Detail Report It is now possible to set the scaling of chromatograms in the Variance Detail report. Scroll Wheel enabled in more dialogues The Scroll Wheel on a mouse now allows you to scroll vertically through the lists in the Assemble Interactively dialogue. Sort Project Windows by column Project Window columns may now be sorted in an ascending or descending order for any column. Improved support for file extensions on Apple s Snow Leopard operating system We addressed the change that Apple Computer introduced so that all the correct file types are visible in the Open Project dialog, including files ending with.spf,.spf,.spr and those with allowed Apple TYPE and Creator codes. Trim Log File There is a new command to assist you in navigating to and opening the log file. More Don t Show Again checkboxes At the request of users, more warning dialogues have been given a Don t show this message again checkbox. Sequence trimming is much faster Ends trimming and vector trimming are now substantially faster operations, especially on large data sets. Gene Codes Corporation 2010 Sequencher Release Notes p. 3 of 13

4 Improved Snow Leopard compatibility (Macintosh) We addressed changes that Apple Computer made which affected file type recognition as well as a change that affected Clustal. Improved File Lists on Macintosh We addressed an issue so that ACE and CAF files are now enabled and selectable in the file lists when importing ACE and CAF projects. In Assemble by Name, more room for Advanced Expression The Advanced Expression type-in box has been made wider to accommodate longer advanced expressions for complex naming conventions. Unusable characters in file names are detected on Windows We catch and handle a problem that was seen primarily on non-english systems on Windows. Unrecognizable characters in the full path name of a file are detected and tell you what needs to be changed for import or export. Lost user preferences have been fixed An issue has been fixed where some of the User Preferences could get lost. One manifestation of this was that you might get asked to register your copy of Sequencher over and over again. % Quality for Contigs A contig will have a % Quality value when Consensus by Confidence is in use. Increased number of bases trimmed in Vector contamination The limit of bases trimmed in Vector contamination has been increased to 99 at each end. Gene Codes Corporation 2010 Sequencher Release Notes p. 4 of 13

5 What s New CONSENSUS BY CONFIDENCE The Plurality and Inclusively consensus calculations are good approximations for creating a snapshot of the consensus of a contig. The default blue shadings for confidence scores of individual bases and the Confidence Histogram have added a finer level of detail of the confidence of individual bases. The new Consensus by Confidence calculation method allows you to calculate the consensus sequence using the confidence scores of the underlying data and then adding a calculated confidence (with appropriate default shading) to the consensus as a user option. Enabling Consensus by Confidence Under the Contig menu is a new item, Consensus by Confidence. Changing to this consensus calculation method will trigger a recalculation of the consensus. Outwardly, this will not look any different from other consensus calculations so ensure that Display Base Confidences is also selected from the View menu. This will confer the color ranges you have set in the Confidence pane of User Preferences on the consensus line. Create New Sequence from Consensus and Confidence Scores If you have used Consensus by Confidence to calculate the consensus of your contig, when you use Contig>Create New Seq From Consensus, the newly created sequence will include confidence scores. CONTIG COVERAGE GRAPH You have always been able to see a representation of the coverage of your contig in the Overview. For users with very high volumes of data, this is sometimes a cumbersome representation of the depth of coverage of your sequences. Now you can see a separate report view representing a scaled schematic of the number of forward and reverse base calls at every position in the contig. Gene Codes Corporation 2010 Sequencher Release Notes p. 5 of 13

6 How to view the Coverage Graph Under the View menu you will find that a new item called Coverage Graph has been added to the Bases, Map, Overview sub-menu. You can enable the Coverage Graph from the Overview, Bases, or Summary views of the Contig Editor. The Coverage Graph will replace any of these views. The graph can be printed by selecting the Print command from the File menu. GENERATE A SHUFFLED SEQUENCE Shuffled sequences are new sequences based on selected sequences or contigs. The bases are randomized to create the new sequences giving you the same composition but a different order. This is a feature that was added by request for use as a statistical control when BLASTing. Generating a Shuffle Sequence Under the Sequence menu, you will find a new item called Generate Shuffled Sequence. To use this item you must first have selected one or more sequences or contigs. For each item selected, a new sequence will be created called ItemName Shuffle 1, where ItemName is the name of the selected sequence fragment or contig and the Shuffle sequence is created by randomizing either the fragment sequence or the consensus sequence of the contig. The newly created sequences will have the same nucleotide frequency as the initial fragment or consensus sequence but different di- and tri-nucleotide frequencies. Note that the sequence created is a DNA fragment. It will not contain any trace data or confidence scores even if these were available in the original sequences. FREQUENCY HISTOGRAM AND TABLES This is another feature that is easily available on the web but was added by request. Nucleotide frequency information is now available as a report. You can generate a histogram and mono, di-, and tri-nucleotides tables for the experimental sequences or consensus sequences you have selected. Creating a Frequency Histogram or Report Under the Sequence menu, there is a new item called Analyses. When you choose this item you will see two choices appear in a sub-menu. The items are Frequency Histogram and Frequency Report. If you choose several sequences and select Frequency Histogram, the frequencies of all the sequences will be plotted on the same histogram. There are a number of locations where you can access these analyses. You can choose a sequence or contig from the Project Window or a sequence or sequences from the Contig Editor and Bases Variance Table. Gene Codes Corporation 2010 Sequencher Release Notes p. 6 of 13

7 The frequency histogram and the frequency tables appear in a new window, which can be saved as a PDF or printed. SUPPORT FOR CONTIG EXPRESS PROJECT FORMAT For those of you who have legacy projects assembled with ContigExpress (file extension.cep), you can now import those projects into Sequencher, including sequence bases, sequence names, contigs, electropherograms, features, confidence scores, base edits (including insertions and deletions), and comments. Importing ContigExpress projects You can now import Contig Express projects using the new File>Import>CEP Project menu command. Sequencher automatically recognizes what a file with the.cep file extension is and imports the data contained therein, including Lite contigs. Fragments and contigs that do not have complete chromatogram data will be imported but minus their chromatogram data. NEW IMPORT FORMATS Sequencher has an extensive number of import formats; to these have been added FastQ files, including the confidence scores and comments, and FastA Aligned. Gene Codes Corporation 2010 Sequencher Release Notes p. 7 of 13

8 Importing FastQ files FastQ format contains sequence data, comments, and confidence scores. There are two main variants of this format: the original/sanger version and the Solexa/Illumina version. Sequencher recognizes and imports both versions. Any file with a file extension of.fq,.fq,.fastq, or.fastq will be recognized as FastQ format. To import these files, use File>Import>Sequences. Importing FastA files Standard FastA files are text files that contain a single sequence. They start with a line which begins with the > symbol followed by some text representing the sequence name. On the second and subsequent lines, any characters will be sequence characters. Concatenated FastA files are similar but contain two or more sequences following each other. FastA Aligned format is difficult to interpret by eye because each sequence in the file has been padded out with symbols so that all the sequences are the same length. These are usually the output of alignment programs. It is important to use the right import method in order to achieve the desired results. If your FastA aligned file contains more then one sequence, choose Import>FASTA aligned from the File menu and Sequencher will create a contig containing all the sequences in that file. If the FastA aligned file is dragged and then dropped onto the Project Window, Sequencher will import all the sequences as individual sequences. CONSENSUS BY CONFIDENCE AND THE VARIANCE TABLE You have always been able to see Confidence Range coloring in a Variance Table created from a single contig using one of the Sequence>Compare Bases To menu items. When the consensus calculation is Consensus by Confidence and the Variance Table is created using Contig>Compare Consensus to Reference, you will now see Confidence Range colors in the cells of the table where there are variant bases. Cells marked with a pink X indicating no coverage will continue to have a white background. DIFFERENCES COLUMN ADDED TO VARIANCE TABLE A new column containing the total number of differences has been added to a number of reports. This has been added to the Variance Table Report as part of the open report, and the Copy as Text and Save as Text export formats. It has also been added to the Individual Variance Reports Open Report output. The Translated Variance Table has also had a Differences column added to its Copy as Text and Save as Text output. Gene Codes Corporation 2010 Sequencher Release Notes p. 8 of 13

9 SORT BY HANDLE Users can now choose to sort the items in a contig by assembly Handle. Sorting by Handle in the Contig Editor Overview window Select the Sort button in the Contig Editor Overview window and the by Handle radio button in the Sort Contig Fragments dialog. The sort orders the sequences in alphabetical order by handle. ENHANCED APPLESCRIPT SUPPORT ON THE MAC AppleScript support has been extended by the addition of a number of new commands to Sequencher s dictionary. Viewing the AppleScript Commands To see what all of the available commands are, look under AppleScript Editor s File menu and choose Open Dictionary. From the list of programs, choose Sequencher to get a list of commands and variables (values) that you can access. You can now set the new Consensus by Confidence calculation method using AppleScript as part of your workflow. How to set the label colors of items You can now get information about the color of a label as well as set the label color of fragments or contigs. In this example script, all the contigs in the project will have their label color set to cyan. You can change the color by replacing the word Cyan in the script with None, Blue, Green, Magenta, Red, or Yellow. How to set the selected state of items If you want to use AppleScript to tell you which contigs are selected in your project, you could use a script similar to this one. The script counts the number of contigs in the project and tells if the contig is selected (true) or is not selected (false). You can use the related AppleScript command set SelectedState of to select or deselect a fragment, contig, or refrigerator. Gene Codes Corporation 2010 Sequencher Release Notes p. 9 of 13

10 How to get or change Comments If you want to use AppleScript to tell you the comments in the Get Info window of fragments or contigs in your project, you could use a script similar to this one. This script will retrieve the comments and display them in the lower pane of the AppleScript Editor. With more complex scripts much information can be written to an external file. What s Improved MULTIPLE FEATURE LISTINGS Your ability to work with Features has been extended by allowing you to open the Feature Listing windows of a number of sequences at the same time. Viewing multiple Feature Listings You can view the information contained in sequences downloaded from GenBank or annotated manually by using the Feature Listing command. You can now view multiple listings by selecting two or more sequences from the Project Window and choosing Sequence>Feature Listing. VARIANCE TABLE DETAIL REPORT CHROMATOGRAM SCALING You can now set the scale in the Variance Table Detail Report for the display of chromatograms. Enabling scaling for the Variance Table Report The default setting for the display scale is 100%. When you have created the Variance Detail Report, click on the Options button. Then choose the % scaling you want to set for the chromatograms in the report. Notice that you can also set the number of chromatograms that can be displayed. Gene Codes Corporation 2010 Sequencher Release Notes p. 10 of 13

11 Sequencher remembers how you prefer to scale your chromatograms for the next time you create a Variance Detail Report. The two images show 75% and 95% scaling of the chromatograms. SCROLL WHEEL SUPPORT You can now use the scroll wheel for vertical scrolling in the Assemble Interactively window. Previously you could only use the sliders for this function. PROJECT WINDOW SORTING Project Window columns may now be sorted in an ascending or descending order for any column using the Shift key. Reversing Window Sort order The Project Window can be sorted using View>Sort/Cleanup and choosing one of the available options from the sub-menu, or by clicking on a column header. You can use the Shift key and click on a column header to sort the column in reverse order. TRIM LOG FILE The Trim Log file contains a record of trims made when the Trim Ends Without Preview or Trim Ends commands are used. This file can be viewed in a text or word processor but is probably best viewed in a spreadsheet. There is a new command that assists you in navigating to and opening the log file. Opening the Trim Log File Choose the menu command Window>Open Trim Log Folder. This will pop up an explorer (Windows) or finder (Mac) window for the Trim Log file location. DISABLING TEACHING AND WARNING DIALOGS Many reminder and warning dialogues become a nuisance once you are familiar with Sequencher. We have increased the number of dialog that show the Don t show this message again check box by adding one to the Trim to Reference Sequence confirmation dialog. Gene Codes Corporation 2010 Sequencher Release Notes p. 11 of 13

12 PERFORMANCE IMPROVEMENTS Several parts of the program have been sped up considerably as part of our development project for higher data volumes. These include Ends Trimming, Vector Trimming, Trim Without Preview, and Import. IMPROVED SNOW LEOPARD COMPATIBILITY (MAC ONLY) We addressed the change that Apple Computer made in the location of the defined temp directory. In particular, this affected Clustal. We addressed the change that Apple Computer made so that all of the correct file types are visible in the Open Project dialog, including files ending with.spf,.spf,.spf, and those with allowed Apple TYPE and Creator codes. MAC IMPORT AND FILE LISTS We fixed an issue on the Mac for File>Import ACE Project and File>Import CAF Project so that ACE and CAF files show up as enabled and selectable in the file lists. IN ASSEMBLE BY NAME, MORE ROOM FOR ADVANCED EXPRESSION The advanced expression input field on the Advanced Expression for Name Parsing dialog has been made wider to allow a longer advanced expression for a complex naming convention to be visible to the user. UNUSABLE CHARACTERS IN FILE NAMES ARE DETECTED ON WINDOWS We catch and handle a problem that was seen primarily on non-english systems on Windows. Unrecognizable characters in the full path name of a file are detected and tell you what needs to be changed for import or export. LOST USER PREFERENCES HAVE BEEN FIXED An issue has been fixed where some of the User Preferences could get lost. One instance of this was that you might be asked to register your copy of Sequencher over and over again. This may recur if you switch between 4.9 and , so it is best to stay with Sequencher once you have installed it. Gene Codes Corporation 2010 Sequencher Release Notes p. 12 of 13

13 % QUALITY VALUE FOR CONTIGS We have extended the display of % Quality in the Project Window from individual fragments to contigs. The % Quality column will contain a value if the consensus calculation is Consensus by Confidence and the calculation is based on the percentage of bases whose confidence score value is not Low. INCREASED NUMBER OF BASES TRIMMED IN VECTOR CONTAMINATION The limit of bases trimmed in Vector contamination has been increased at each end. You can now move the spin control in the Vector Trim Parameters up to a maximum value value of 99. Gene Codes Corporation 2010 Sequencher Release Notes p. 13 of 13