Using BioEdit

BioEdit is a freeware for editing alignments of nucleotide or amino-acid sequences. It can display traces of ABI sequence files.
It is available at;
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
Current version is 7.0.9.0.
The package can be installed through the standard Windows procedure. Commands are available on the menu-bar, and it may be easy to learn commands just trying and using it. I felt, however, difficulties using it at the beginning, because (1) behaviors related to select and copy are sometimes different from the standard Windows action, (2) menu items in depths is difficult to find out, and (3) usage of special functions is difficult without tutorials.
A simplest tutorial of the BioEdit is as follows.

Non-Standard Actions Related to Select and Copy
Users may feel difficulties using the BioEdit because of its particular behaviors related to select and copy. Learning the non-standard actions, it becomes easier to use it.

Behavior of BioEdit ver.7.0.4.1 related to "select and copy". Actions different from the windows standard are written in red. 
Mode/Action Click on a residue Shift+click on a residue Edit->Select to End Edit->Select to Beginning Drag residues with the mouse left button on. Drag ruler with the mouse left button on. Sequence->Select Positions, upon a region of other sequence selected. Edit->Copy Sequence(s) Edit->Delete Sequence(s)
or
Cut sequence(s)
Select/Slide Highlight the residue to select. Expand the selection on a line or a square area.
Clicking on the opposite side, expand to the both direction.
Unable to diminish.
Select to the end including the current residue.
Indication of selected region on the aligment window not changed.
Select to the beginning including the current residue.
Indication of selected region on the aligment window not changed.
Select a square region. Select all residues of all the sequences. Select both regions. Be careful to copy. Copy the data of highlighted sequence.
Not copied to the system clipboard.
Paste Sequence(s) command pastes the sequence title and residues.
Delete (and copy) the data of highlighted sequence.
Overwrite the sequence title onto the next title shifting up, when the title is being edited.
Edit Move cursor between the residue and the previous residue.
Unable to copy.
Changing mode from others, the cursor moves one residue before.
Expand the selection on a line or a square area.
Clicking on the opposite side, expand to the both direction.
Unable to diminish.
Select from the next next residue to the end.
Indication of selected region on the alignment window not changed.
Select from the next residue to the beginning.
Indication of selected region on the alignment window not changed.
Select a square region.
Move cursor between the top of the selected region and the previous residue.
Select all residues of all the sequences. Select both regions. Be careful to copy. Copy the data of highlighted sequence.
Not copied to the system clipboard.
Paste Sequence(s) command pastes the sequence title and residues.
Delete (and copy) the data of highlighted sequence.
Overwrite the sequence title onto the next title shifting up, when the title is being edited.
Grab&Drag Highlight the residue to select. Disabled. Select to the end including the current residue.
Indication of selected region on the alignment window not changed.
Select to the beginning including the current residue.
Indication of selected region on the alignment window not changed.
Disabled. Select all residues of all the sequences. Select both regions. Be careful to copy. Copy the data of highlighted sequence.
Not copied to the system clipboard.
Paste Sequence(s) command pastes the sequence title and residues.
Delete (and copy) the data of highlighted sequence.
Overwrite the sequence title onto the next title shifting up, when the title is being edited.


Commonly used menu items for editing DNA sequences
When I sort out sequence regions in a long stretch of sequence, I save the following four kinds of files to make alignments for phylogenetic analysis.
(1) The original. Contig sequence exported from consed (FASTA format) or reference sequences downloaded from databases (GenBank format).
(2) Working file. Multi-FASTA format file containing the original and sequences regions of interest sorted out from the original. Files handled with BioEdit can contain a number of sequences and files are called "alignment", but these sequences in a file are not necessarily homologous. The "alignment" file is a file in which a number of sequences can be put together temporarily for editing procedures.
(3) Annotation file. Use a text editor. Sequin can also be used, if you are going to submit the sequence data.
(4) Alignment file. It contains aligned sequences of each region sorted out from the original. It is an input file for phylogenetic analysis softwares.

A simplest editing procedures may have searching regions of interest in the original upon homology search or virtual translation, etc, saving a work file containing the regions of interest, editing annotation file with a text editor, piling up an alignment file of each region, and align sequences with ClustalW or manually. To make the original file, select FASTA format to export consensus with consed with suffix of ".fas" or ".fasta". For downloaded files from DDBJ/GenBank, put ".gb" as suffix. BioEdit recognize both formats with correct suffixes.
These procedures need the following commands among the menu items.
  File->Save or Save As
    Save alignment file. File name and format (FASTA, DDBJ/GenBank, Phylip, etc) can be changed on Save As command.
    NEXUS format is not supported.
  File->New Alignment
    Make a new alignmant file.
  File->Open Alignment
    Open an alignment file (FASTA, GenBank, etc). Some of Phylip format file are not properly loaded.
    Comment line of FASTA format file beginning with ";" makes error.
  Edit->Cut, Copy, Paste, or Delete Sequence(s)
    Copy and paste sequence title and data of highlighted sequence(s) clicking the sequence title(s).
  Edit->Unselect All Sequences or Residues
    Release from selection.
  Edit->Search->Find or Find / Replace
    Search and replace character strings.
  Edit->Search->Search for user-defined motif
    Search for sequence with wobbled sites.
    Search Residues
    Check "Nuceic Acid" or "Amino Acid", then IUB code is accepted.
    Target sequence should be UPPER CASE. Wobbled recidues in the query should be UPPER CASE.
  Sequence->Manipulations->lowercase or UPPERCASE
    Convert the selected sequence(s) or the selected nucleotides to lower or UPPER cases.
  Sequence->Nucleic Acid->Reverse Complement
    Reverse complement the selected sequence(s).
  Sequence->Nucleic Acid->Restriction Map
    Search restriction sites of the selected sequence.
  Restriction Map Maker
  Push [Select from list] and select enzymes for which restriction map is made. Push [Generate Map] to do.   Restriction Enzyme Selector

  Sequence->Nucleic Acid->Translate->Frame 1, 2, 3, or Selection
    Virtual translation of selected sequence(s) of each frame from the beginning or selected region.
    Displays nucleotide and amino-acid sequences altogether. The result can be saved as a text file.
  Sequence->Toggle Translation or Toggle translation in selected frame
    Virtually translate and display the amino-acid sequence replacing with the nucleotide sequence in the alignment window.
    The amino-acid sequences can be paseted to other sequences or alignment files.
    The original nucleotide sequences are retaind even if saved to overwrite a file. Use this command again to revert.
    Translate.. (permanent) replaces the original nucleotide sequences with the translation permanently (unable to revert).
  Accessory Application->BLAST->Local BLAST
    Homology (BLAST) search to a local sequence database constructed on the HDD of your PC.

    Local BLAST

    Paste the query sequence onto the sequence field, or automatically pasted upon clicking the sequence title to highlight.
    Displays query and subject (database) sequences altogether, and can be saved as a text file.
    To search for fish mitochondrial tRNAs, for example, adjust
      -q  Penalty for a nucleotide mismatch
      -r  Reward for a nucleotide match
    parameters to, say, -2 and 3 respectively (input "-q-2 -r3" in the field of Additional parameters). Then the result may be better.
  Sequence->New Sequence
    Add new blank sequence to the alignment file.

    New Sequence

    A window of the blank sequence opens, and paste the sequence data and the sequence name.
    Also for existing sequence the sequence window opens upon double click on the sequence title.
    Click on the red arrow at lower right corner of the window, then edit fields for annotation appear, but it is awkward to use.
    It is better to draw a related sequence from DDBJ/GenBank and use it as a template for editing annotation with a text editor or Sequin.
  Accessory Application->ClustalW Multiple alignment
    Automated multiple alignment with ClustalW.
    Sometimes the FAST algorithm may work well with sequences of low homology with many gaps.

Special Functions
  Accessory Application->BLAST->Create a local nucleotide database file
    Construct a local nucleotide database on HDD of a PC (local).
    Save a multi-FASTA file containing all the sequences to be included in the constructing database in the ".\BioEdit\database\" directory.
    For an example data of search for flatfish and cypriniforms mitochondrial tRNAs, click on either.
    Select the FASTA file on this command. Then a DOS window opens and a database construction program runs.
    After construction of a database, it becomes available for the Local BLAST command.
  Options->Select Codon Table
    Change codon table from the universal.
    Selected codon table can be displayed with Options->View Codon Table function,
    but the new codon table is activated only after restart of the program.
    Prior to use this command, save a codon table file with a suffix ".tab" in ".\BioEdit\tables\" directory.
    For vertebrate mitochondrial codon table, click here.
  View->Customize Menu Shortcuts
    Configuration of short-cut keys to bypass the menu.
    It rewrites ".\BioEdit\BioEdit.ini". I customize the short-cut key menu as follows.
      Save As...=Shift+Ctrl+S
      Close=Ctrl+W
      Exit=Ctrl+Q (This shortcut is disabled unless file is opened)
      New Sequence=Shift+Ctrl+N
      Select Positions=Ctrl+L
      Unselect All Sequences=Ctrl+U
      Unselect All Residues=Shift+Ctrl+U
      Select to End=Ctrl+E
      Select to Beginning=Ctrl+B
      Find / Replace=Shift+Ctrl+F
      Search for user-defined motif=Ctrl+Alt+F
      Toggle translation in selected frame=Shift+Ctrl+G
    Carefully set short-cut keys to avoid duplicate definition. Sub-menus in the depths of the nested items seem to unable to set.
    I further customize the file mode as editable & insert as follows.
      [Default Mode]
      Mode=1
      Overwrite Mode=0
    On this configuration, ABI traces (electropherograms) become editable, but base insertion and undoing are unable (revertable, though).
  Options->Preferences
    Change other settings. For example, set translate command to show selected regions only and one-letter code for amino-acids.

    ORF Setting



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