Overview: The core of the display is a standard text alignment of all of the residues in the sequences aligned. Amino acids are shown with the standard one-letter code and indels (gaps) are denoted by dashes ("-"). Ticks and numbers for the alignment are shown above the alignment. Residues are shaded to indicate the quality of their match score, with darkest blue being the best, white being zero, and pink being all negative scores. The specific score for any residue can be gotten by allowing the mouse to hover over one of the residues. Often a more realistic sense of local alignment quality can be gotten by viewing a score that averages across local residues (the score window). Many display settings can be made from the Edit, View, and Show menus. You can change the order of sequences in the alignment by highlighting a sequence or block of sequences (click in the name panel) and using Cntrl-up and Cntrl-down.
Score Window: This setting controls how many aligned residues are averaged to produce the score shading (and number). The initial display is set to one, meaning that the score shading for each residue reflects the score quality only at that single position. Larger score windows cause each position to be assigned a score based on the average of it and residues around it. Try increasing the score window to see how the quality "blurs" out as the window increases.
Selecting Residues for Realignment: You can realign any part of the current alignment by highlighting the residues and using the menu "Align : Align Selected Columns". You can use this to manage easily-viewed regions of an alignment, to trim sequences to shared conserved regions, or to change the alignment parameters for comparison. Use the menu "Settings : Multiple Alignment Settings" to bring up the standard Multiple Alignment Settings dialog, which can be used to change these parameters. Changes you make here will not affect the settings in the main Bonsai window. Note: Inferred phylogenetic relationships will often change based on what parts of the sequences you align - if you are interested in the evolutionary origin of the core shared sequence among a set of proteins, you must align only their core shared regions.
Interpreting Alignment Quality: The color coding and the numerical scores are calculated by sum of pairs and indicate how well the residue in question matches the other residues in its column, taking account of residue similarity through a score matrix. [Explanation should be expanded]
Notes on Alignment Quality: The alignment shown is guaranteed to be one best-scoring alignment for these two sequences given the assumptions implicit in the alignment model (see Score Matrix Help for specifics). However, there is NO GUARANTEE that there aren't other alignments that are very close to this one in score, or even equal in score. Unless the sequence has unusual features like repeats with high similarity, these other alignments will be relatively modest variants on this alignment, but they can be highly significant in local regions of the alignment.
Save Alignment: Save this multiple alignment in one of several text formats or a Bonsai serialized alignment. The serialized form is convenient in some ways but is NOT guaranteed to be stable in subsequent releases of Bonsai.
Save Sequences: Save the sequences that back this multiple alignment in Bonsai serialized format.
Save Fasta Sequences: Save the sequences that back this multiple alignment in Fasta format.
Save Selected Columns as Sequences: Save only the residues in the selected columns as sequences in Bonsai serialized format.
Save Selected Columns as Fasta Sequences: Save only the residues in the selected columns as Fasta sequences. This is a convenient way of saving end-trimmed sequences from alignments.
Save as GIF: Save this multiple alignment as a GIF image file. The image will be formed by the sequence name panel and the sequence panel (with tics and numbers) combined, with all other parts of the window removed. It is usually a good idea to convert to multiline display before saving as a GIF.
Print: Print this multiple alignment. Imperfectly implemented: the current version does not print sequence names or numbering and it does not attempt to avoid splitting blocks across pages. Use "Save as GIF" to manipulate and print with a proper image-processing program.
Remove Selected Sequences: Remove the selected sequences from the alignment window for viewing purposes. This cannot be undone! The sequences will also be removed from the backing sequence list and the ability to view intermediate alignments will be abrogated. Except for producing figure outputs, it is best to delete sequences before alignment.
Column/Row/Alignment-Edit Selection Modes: Toggle between mouse selection of rows (e.g. useful for removing sequences), selection of columns (e.g. useful for selecting subsections of sequences for saving or realigning), and multiple alignment gap-editing. Multiple alignment editing is only partially developed - see Editing a Multiple Alignment.
Clear Selections: Clear the current sequence or column selections.
Move Selections Up/Down Table: Move a block of highlighted sequences up or down in the view order. Applies only to Row Selection Mode.
Find in Alignment: Find specific residue sequences in the alignment.
Single/Multiple Block Display: A multiple alignment initially displays on a single line for each sequence. This menu item toggles between this mode and showing the alignment in multiple blocks. In multiple block mode, several features are disabled because they are a pain to program [I may fix this]. Make changes in single block display first, then switch to multiple block.
Score/Hydrophobic Color: The Bonsai default is to show the score quality colors. Use this menu item change shading to degree of hydrophobicity.
Increase/Decrease Score Color: Intended to tune the output visibility for different monitors and projectors. The background color intensity changes. This doesn't change anything about the alignment itself or the scores, but it will be reflected in the colors when saved or printed.
Show/Hide Negative Color: The Bonsai default is to show all scores of zero or less in white. This menu item toggles between this mode and showing negative scores in light red, which can be a useful warning for poor alignment regions.
Show/Hide Gap Color: The Bonsai default is to hide colors for gap residues as they can be distracting (for example, a column with mostly gaps shows these as scoring highly!). Use this menu item to show the computed colors for gaps.
Show/Hide All Color: The Bonsai default is to show the score quality colors. Use this menu item to remove or reinstate the colors.
Residue Number/Alignment Score Tooltips: The Bonsai default is to show the residue number when the cursor hovers over an alignment residue. Use this menu item to toggle between this mode and one in which the local alignment score for the residue is shown. Regardless of this setting, the color will reflect score.
Show Menu:
Show/Hide Multiple Alignment Comments: You can display and edit comments on this multiple alignment in a pane that will appear at the bottom of this window. If you use and then close the comments pane or if you have made comments and then save the multiple alignment, changes will automatically be saved. As with sequence lists and individual sequences, these comments will only be saved in the Bonsai serialized format - they are not compatible with standard text alignment formats.
Text Alignment: Show a new window with the text alignment version of this multiple alignment. The same text version can also be shown by checking "Display Text-Formatted Alignment" in Multiple Alignment Settings before carrying out the alignment.
Boxshade: To view the alignment in the more familiar Boxshade format. Useful mostly for black and white figures. Note: there are web-based Boxshade servers that produce better output (e.g. http://www.ch.embnet.org/software/BOX_form.html). They are compatible with the Bonsai text alignment format without residue numbers. Use "Show : Text Alignment", select the entire text alignment, and paste it into the Boxshade server form. Use the "ALN" or "Clustal" format option.
Column Alignment Values: Show a window that displays and permits editing of the Position Specific Score Matrix (PSSM) for this multiple alignment.
Alignment Set Order: You can view much of the history of a progressive multiple alignment starting here. When activated, a number and letters will appear to the left of the sequence names. The number reflects the currently viewed alignment set and the letters reflect the sets of sequences in Group A and Group B for that set (see Multiple Alignment Methods for more). See additional View Menu items for how to get more information from these sets.
Next/Previous Alignment Set: Move up or down through the list of alignment sets that built up this progressive multiple alignment.
Alignment Set Summary: Use to view more about the current alignment set. The panel that displays permits you to view many details of that alignment set and the intermediate alignment it produced, including the score matrix, sequence weights, and alignments themselves.
Progressively Refine: Refine the multiple alignment by extracting one sequence at a time and realigning it with all the remaining sequences. Useful for unfreezing unfavorable local alignments created as an artifact of standard progressive alignments. In cases of very diverse families, this step may make a large improvement. For reasons I have yet to figure out, occasionally this method degrades the alignment slightly. Keep an eye on whether the alignment score improves.
Align Selected Columns: Realign all the sequences using only the selected residues. You must be in Column Select mode for this to be active.
Realign Full Sequences: Realign from scratch all the sequences in the backing sequence list, using the current Multiple Align Settings. Useful if you delete sequences and want to realign the remaining ones.
Align Sequences to This Multiple Alignment: Align the sequences currently in the Bonsai main window with this multiple alignment. You can also do this in reverse from the main window by opening a saved multiple alignment from a file.
Align an Alignment to this Alignment: Align a multiple alignment to this multiple alignment. You will be prompted to load the second multiple alignment from a file.
NJ Tree From Multiple Alignment: Use this multiple alignment as a guide for generation of new pairwise distances and recalculation of a new Neighbor-Joining Tree. Will usually produce a more accurate tree than the original guide tree.
Hydropathy Plot: Calculate a Kyte-Doolittle hydropathy plot for the multiple alignment. The hydropathy values reflect the mean for each column in the alignment.
Pair Alignment Settings: Change the pair alignment settings that will be used in further alignments starting from this window.
Multiple Alignment Settings: Change the multiple alignment settings that will be used in further alignments starting from this window.
Tree Settings: Change the settings for the guide tree that will be used for the realignment.
Multiple Alignment Help: This help.
Understanding Multiple Alignment Methods: A primer on how multiple alignments are constructed, including various methodological and computational issues that arise.