SEQtools contains various options for controlling program behavior grouped
under the Preferences menu of the main editor form.
When you exit SEQtools, the settings are saved in an ini-file located in the
application folder as a plain text file. Each instance of SEQtools gets its own ini-file
making it possible to set different preferences for different instances of SEQtools. For
example you can set different sequence color pattern and background color for instances
for handling nucleotide, protein and primer sequences. Read more
aboutcommand line optionsin
section 3.3 Features of the manual.
It is recommended that you go through and set program preferences as you prefer the first time you launch SEQtools after installing the program. Pay special attention to settings for internet connection and the NCBI ini-file. The latter setting varies depending on your operating system. (Windows2000 or Windows XP)
The different groups of preferences are described in some detail below. Quite a number of user questions relates to problems caused by incorrect preference settings so please read this section carefully.
The eight tabs of the general settings form are displayed below. In most cases the effect of changing these
settings are self explanatory.
184.108.40.206 Project Files - Enables auto-saving plp and psp files each time a project is saved. This implies that you can re-open the project either with the sequences originally loaded into the project (*.plp) or with the files included
after editing and saving the project (*.psp).
220.127.116.11 Colors and fonts - SEQtools requires that you use fonts with fixed character-spacing such as Courier New or Monospace (included in the SEQtools setup file). Using fonts with variable character width will cause the sequence to be distorted when displayed in the editor and in the various sequence lists. In most cases, however, SEQtools uses hard coded font which cannot be changes by the user.
18.104.22.168 Launch Application - This tab allows you to populate the Launch menu of the main editor with up to five applications which will be launched by clicking the menu item. To add an application to the list place the cursor on the line where you wish the application to appear. Click Browse and navigate to the exe-file of the application be to included. Clicking the exe-file places the full path in the left field and the title of the exe-file in the right. Edit the title of the exe-file if you wish.
22.214.171.124 Launch URL - On this tab you can edit the default list of web resources: Type the url of the website in the left text field and the title of the site in the right field. Maximum number of urs is 10. URL incuding the word NCBI will be identified by an NCBI icon in the menu item, other url with a globe.
126.96.36.199 Backup Settings - Setting the interval between timed backups to a positive value specifying the number of minutes between auto-backups activates the auto-backup function. It is not recommended to use this function when you are working with large projects (> 300 sequences). SEQtools will notify you if the size of the projects exceeds 300 and offer to turn off auto-backup.
Note that the batch blast function - which may run over several days - has a separate function for timed saving rearch results to avoid loss of data in case of power failure during the batch job. This tab is also included in the Project Settings form.
188.8.131.52 Checksum Calculation - This tab controls the checksum function. You may gain some speed improvement by turning this function off.
184.108.40.206 DOS Directory - By default the special directory for the various external programs used in SEQtools to perform certain tasks is the one given in the tab below. It is possible - but not recommended - to change this path. I have not tested the effect of placing the dos folder on a different drive and any experiments is at your own risk.
If you change the path SEQtools moves the content to the new destination when you click Accept.
220.127.116.11 Footnotes - In some cases it may be convenient to include a footer when you save a sequence file.
The settings included in this form affect various aspects of the current sequence. .
18.104.22.168 Project Titles - This tab is displayed before loading of sequences into the project commences. It furthermore includes a checkbox activating validation of sequences while loading. This option which is only relevant when you are loading non-SEQtools formatted sequences slows the loading process somewhat down.
22.214.171.124 User Data - This tab enables you to personalise your project by entering your name and e-mail address.
126.96.36.199 Project Files - The tab allows you to set the display preferences for sequence formatting, i.e., whether or not to use upper, lower or both cases; to allow spaces in the displayed sequence and to set line and block length and line spacing.
188.8.131.52 Color Patterns, nucleotide - You can change the color of the nucleotides in nucleotide sequences.
184.108.40.206 Color Patterns, protein - You can change the color of individual residues in protein sequences with the options included in this tab. Note you can selectively highlight amino acids according to their properties with the Basic, Hydrophilic, Acidic and Hydrophobic options.
220.127.116.11 Color Patterns, primer - You can change the color of individual residues of primer sequences with the options included in this tab. Note that you can selectively highlight degenerate positions in the sequence with the IUB = Red option.
18.104.22.168 Header - Note that thecompositionof the sequence header is controlled by a separate set of quite extensive options which allows you to precisely select which part of the total sequence annotation you wich to include in the displayed header.
22.214.171.124 Trace File Folder - This tab allows you to set the path to the folder containing the chromatograms from which sequences in the project are derived. The path is automatically set to that of imported/basecalled chromatograms (=trace files) when they are loaded. If, however, you import chromatograms located in different folders it is necessary to set the path to the folder containing the underlying trace files. Obviously it is preferrable to place all trace files to be included in a project in the same folder.
126.96.36.199 Global Timeout - SEQtools uses a number of external programs running under the dos operating system. To do this SEQtools creates a "memory space" to hold the process and monitors the process to detect when it ends, i.e., when the task is completed. If something goes wrong and the externat program fails to issue the completed signal the process will run for ever unless a timeout is set to kill the process if no response is received within a certain period of time.
If everything works as it should the timeout value has no effect as long as it is sufficiently large to allow external programs to complete. If, on the other hand, the problem is temporary, a long time out value may allow the halted process to resume and run to completion. You need to experiment to find the optimal value. Sixty seconds works fine in most cases.
188.8.131.52 Project Blast Settings - Pressing <F5> will create a local database containing all sequences on the project and perform a blast search on this database, i.e., perform a Project Blast. The result of this project blast search is displayed in the sequence list in the right hand panel of the main editor (on a light blue background).
The settings in this tab controls the number of matches to display and an expect value cutoff. Setting the maximum number of matches to zero turns off this function (which there is no reason to do).
184.108.40.206 Backup Settings - Setting the interval between timed backups to a positive value specifying the number of minutes between auto-backups activates the auto-backup function. It is not recommended to use this function when you are working with large projects (> 300 sequences). SEQtools will notify you if the size of the projects exceeds 300 and offer to turn off auto-backup.
Note that the batch blast function - which may run over several days - has a separate function for timed saving search results to avoid loss of data in case of power failure during the batch job. Note that a Backup Settings tab is also included in the General Settings form.
SEQtools uses separate forms for most tasks. This often results in the screen being crowded with forms -
the one you which to use in most case being hidden behind other forms. To give you the option to excert some control
of the form behavior the settings in the Form-Stay-On-Top preferences may be useful. By putting a checkmark in
the box for the form the form will remain at the top position, even when it hasn't got the focus.
Setting these preferences requires some experimenting...
One of the strong features of SEQtools is the functions for customising sequence listing to facilitate
getting an overwies of the sequences contained in the project.
The four tabs included in Description Line Format settings are describes below.
220.127.116.11 Format Settings - The check boxes on this tab enable/disable different formatting options. In addition it is possible to set a cutoff expect value to exclude blast results with worse than the specified match from the sequence list.
The File menu includes save and load options for the selected formatting scheeme to allow you to easily switch between different formatting scheemes. Line formatting scheeme files (*.lfs) are saved in the DataFiles folder of the main SEQtools application folder. A few *,lfs files are included in the setup file to illustrate how quite detailed line formatting can be acomplished with the different line formatting options.
18.104.22.168 Left-Trim Lines - With this option you can enter a list of words to be removed from the start of the description lines. Removing occurs sequentially from the top of the list.
Assume for example that a large number of the description lines have the word Rat as the first word hypothetical as the second word. Adding Rat as the first word in the list and hypothetical as the second will the remove both words from the description lines. Whereas the two words in reverse order will remove nothing from such lines.
In some cases you may want to remove everything from a specific character. This is possible by adding "<-" in front of the character from which you want to truncate the line. The"()" code removes the first word in brackets whereas "-()" removes only the brackets. Single characters can be removed by adding the character in leftmost column.
22.214.171.124 Right-Trim Lines - This tab includes the same options as the Left-Trim Lines only the formatting occurs from the right end of the lines.
126.96.36.199 Replace Lines - In many cases the best match of a blast search returns a description line indication that the match is to a hypothetical, putative, similar to, deduced or a probable protein. This is not very helpful if you are looking for an indication of a possible function of the protein.
By using the Replace Line filter you kan make SEQtools skip description lines containing the forbidden words and instead look for alternatives further down the blast results - but still within the expect value cutoff set at the first tab of this form.
If SEQtools is unable to find a description line without one of the forbidden words and within the expect value limit the first description line (=best match) is returned.
The behavior of ConvertTrace is handled by SEQtools and do not require any user
intervention. The format of trace files to be imported is automatically detected by SEQtools and passed on to
ConvertTrace. ConvertTrace accepts most common trace file formats including ABI, ABD and SCF.
The SEQtools interface to LifeTrace on the other hand gives the user extensive control over the basecalling process as well as of the removal of low quality regions from raw sequences. Read more about basecalling with LifeTrace under the File menu.
188.8.131.52 Fixed scheemes - The SEQtools user interface to LifeTrace currently includes three fixed schemes for basecalling and trimming trace files (sloppy, standard and stringent), all based on:
184.108.40.206 Options for Basecalling - Setting optimal parameters for basecalling with LifeTrase is not trivial and may require some experiments before the best result is achieved. Useful hints are described below.
The currently active parameters for basecalling and trimming are shown in a text box on the Preset Options tab.
To compensate for differences in signal strength between individual sequence runs, an option is
included to slightly modify the parameters compensating for varying signal strength among different trace files.
With this function, Adaptive N-Threshold, enabled qscores are adjusted in the following way:
QualityScore = QualityScore + ((MeanScore - NeutralMean) / 15).
As can be seen from the equation above, this adjustment has no effect when the mean of all quality scores for the trace file is equal to the 'neutral mean'. For mean scores greater than the 'neutral mean', quality score cutoffs are increased while score cutoffs smaller than the 'neutral mean' score are reduced.
This option allows you to introduce a slight adjustment of the set parameters to compensate for small variations in the overall quality of individual trace files.
This option as well as the quality scores, window sizes and gap quality scores currently selected for the three fixed settings may be adjusted if suggested by further testing of the function.
220.127.116.11 Trimming Raw Sequences - Based on qscore values and a set sliding window length it is possible to perform a quite precise trimming of raw sequence files. Activate the adaptive N-threshold option to reduce the effect of varying peak height in the chromatograms.
18.104.22.168 N-Threshold - Options to control when the basecaller inserts an N when the basecalling is ambiguous. Activate the adaptive N-threshold option to reduce the effect of varying peak height in the chromatograms
22.214.171.124 Gap-Quality - Finally it is possible to have flanking, dubious bases displayed in lower case when the peak spacing is irregular (gap_qscore). This information is relevant to highlight regions where the number of bases within a short region of the sequence cannot be determined precisely.
NCBI Ini-File - Using the various programs provided by NCBI require that a NCBI ini-file is present in the Windows folder. The ini-file contains the path to the NCBI data folder. Depending on your operation system, the name of the Windows folder is WINNT (Windows 2000) or Windows (Windows XP). This difference must be incorporated in the NCBI ini-file. In case you are behind a firewall you may also have to edit this information in the NCBI ini-file.
The NCBI Ini-File editor include relevant information and options for these tasks.
126.96.36.199 Internet Connection - SEQtools verifies the internet connection each time the program is launced. This is done by connection to the Google server at the URL http://www.google.com/. It is possible to change this URL if you wish to test the connection to a different server.
188.8.131.52 NCBI Resources - The NCBI Settings tab includes URLs to various NCBI resourcers. These values should not be changed by the user (unless you known exactly what you are doing).
The default project restriction enzyme data file can be composed using the enzyme group editor shown below. The first tab includes a number of fixed patterns while the second tab allows you to create a highly specific enzyme file.
After clicking Accept the selected enzyme file will be the default until it is changed.
Several log and data files are created by SEQtools or by the associated external programs. Often it is difficult to locate such files on your hard dish. This form includes links to all program generated text files.
It is possible to run parallel instances of SEQtools. This can make it quite confusing/difficult to identify to which instance a particular form belongs. With the Color Coding option you can choose an identification color for each instance of SEQtools. This color is visible on most forms derived from the color coded insance.