Tom Hall. North Carolina State University, Department of Microbiology. This is likely to be the final release of BioEdit. There may be some bugs. BioEdit is a mouse-driven, easy-to-use sequence alignment editor and sequence analysis program designed and written by a graduate student. BioEdit can also edit chromatograms, but I find Chromas to be nicer. MEGA also has an alignment editor, but I’ve not really used it very much. Double click on the .
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Chromas has the advantage the you can save all of your chromatograms which can subsequently be used in any other programs unlike Sequencher which saves everything in a project file which cannot be opened by anything else.
If I loose my sequence alignment, at least all my chromatograms with the correct edits are still there to rebuild it from. BioEdit can also edit chromatograms, but I find Chromas to be nicer. Double click on the chromatogram file usually has the extension ab1.
This opens the file in Chromas see below under installation notes if some other program opens it instead of Chromas.
The chromatograms come off the machine with all bases in upper case. I usually make all of my edits as lower case bases as it makes it easier to identify where I have made edits. When I am done I save the chromatogram and export the data to a line file which is saved with a. One trick I find useful later is to always edit your sequences from the same starting base unless the starts are all messyas it makes sequence alignment much easier later. I use BioEdit to align sequences as it is free and has some handy features.
The most annoying aspect is that you have to manually align up each sequence and manually create a consensus sequence which commercial programs like Sequencher and Geneious are very good at. It is the only program I know of that allows you to edit, search and replace, and paste over the sequence title names independent of your sequences.
I use this feature on nearly every dataset I create. As far as I can tell there is no difference between saving your file as a BioEdit formatted file versus as a fasta file. One quirk of BioEdit is that if you double click a data file it will open in a new copy of BioEdit, not in an existing one.
The regular copy and paste features work between copies of the program, but copying and pasting sequences does not. I then select those sequences control-shift-acut control-shift-c or copy them control-a and paste them control-s to the desired BioEdit file.
The reason why I paste them to a new file first is that importing from the clipboard File, Import from Clipboard will place them at the bottom of your file, which is usually not where I want them be. Once I have edited all of my chromatograms I copy the. Open BioEdit from the start menu. Note that I have changed or set many menu short cuts see BioEdit stuff to change after installation below to make things quicker, thus these instructions are based on these changes.
Create a new BioEdit file. If you wish to keep them in the same order as they are in your directory then click on the bottom sequence file first, then click on the top one while holding the shift key. Make sure your mode is set to edit and insert. It helps if you edit the sequences to start from the same base prior to importing them, that way if you do multiple sequences they are already mostly aligned.
There is no auto save function. I usually import all the forwards and reverses into a new BioEdit file. I first group all the forwards together, then all the reverses. I manually align them and check for obvious missing bases and either correct them or add a gap to preserve the alignment.
Before trying to merge the forwards and reverses together, reverse the first reverse sequence Sequence, Nucleic Acid, Reverse Compliment or control-shift-r and align it to your forward sequence usually I have to delete a few bases. Then reverse compliment all of them and they should be perfectly aligned relative to the forwards. Note that this works best with coding sequences without indels as every sequence is an identical length, it is all a bit trickier with different length sequences.
In that case I try and get them close, but each individual one many require adjustment. Once I am happy with that I ready to create what will become the consensus sequences. I copy all the forwards to a new BioEdit file, select the sequence titles Edit, Select All Sequences, control-shift-a and copy them to clipboard Edit, Copy Sequences, control-amake the new BioEdit file active and paste them in Edit, Paste Sequences, control-s.
I copy the sequence titles to the clipboard Edit, Copy sequence titles. I paste these into Microsoft Word and use search and replace to get rid of extra details. My sequence names look like this, PU Glu31 and replace with. Select them all control-acopy to clipboard control-cgo back to BioEdit, to paste these names over the existing ones.
Go Edit, Paste Over Titles. Now your BioEdit file has all the forwards and reverses, with the. It helps to also have additional individuals from the same population all next to one another too. To correct the consensus sequence I copy and paste the sequences from a population or individual, group, etc.
Change the view type on the lower toolbar 3rd of the alignment windowselect the third colored button from the left says Shade identities and similarities when you hold the mouse over it.
This highlights any columns that have different bases. Depending on how well your reverse sequences overlap with your forwards, scroll right until they overlap with good sequences. Select all the reverse sequences and cut them. This will allow you to see any base pairs that are different in the clean forwards.
I check any unique differences by opening the chromatogram. Now scroll right again and look for any bases that need tuhorial. Eventually the forwards will start to be a poor match to the reverses.
Guide to editing sequences with Chromas and BioEdit
boiedit At that point I finish my consensus sequence. I select a point in the reverse, then select sequence to the end Edit, Select to End, control-e.
Now place the cursor in the same place in the consensus sequence. Hit control-e to select to the end, hit delete, move right one base then paste control-c. Repeat for each consensus.
Just be sure to select to end from a different location each time to reduce the biordit of pasting bioedt wrong reverse into your consensus. Now I select all the forward sequences and cut them and scroll right to check for any bases changes that need to be checked. Then I undo the cut, select all the sequences Edit, Select All Sequences, control-shift-acopy them control-a–note that copy and pasting sequences is different to any other copy and paste action.
Go back to your BioEdit file with all your sequences which should still have the original sequences highlightedpaste the sequences control-sthen delete the selected sequences control-dthus replacing the newly edited ones bioedkt removing the originals. Hit save control-shift-s and repeat for each group of sequences.
Sequence editing using BioEdit
At the end of this phase you have done two data checks, one when you edited your original chromatogram, second when you checked any unique base pair changes.
For each gene within a dataset I usually have this file with the forward, reverse and consensus. I then create a second file which has only the. I always keep the BioEdit file with all forwards, reverses and consensus sequences so that if I double check stuff later it is easier to find the relevant chromatograms I can tell what sequence is from where by the sequence name.
All of that probably sounds very confusing, once you have carefully worked through it a couple of times it becomes very easy. In BioEdit, clean up all the ends and get things to the base pairs you want to analyze. It can be helpful to make sure any missing bases are labeled with an n, only use a – for indels so that you can easily distinguish which is which. Remove the existing sequences from the first sequence hit control-shift-end, then hit deletethen paste in the ones you just copied.
Guide to editing sequences with Chromas and BioEdit
Note how many replacements it does, this is the number of samples. Enter that information in the header of the MEGA file. Figure out how many base pairs are present in BioEdit, go to the last base and select it and look at the number.
Save the file as text only and make sure it has the correct file extension. If the program sticks the. Then I run a NJ analysis to see what is going on with the dataset. When you first install BioEdit and Chromas, the default will be that BioEdit opens the chromatogram files. To fix this, right click on a chromatogram, select properties, it should say opens with BioEdit, hit change, browse to the Chromas executable, select it, choose always open with this program, hit ok.
Now when you double click on a chromatogram it will open in Chromas. BioEdit lets you modify just about anything that it does relative to menus and keyboard short cuts as well as the default settings for displaying data. Once you set your preferences on one machine you can copy the bioedit. You can download my bioedit. These are my preferences, you can use these or change them whatever you prefer.
I hate menus, so anything that I can use the keyboard for I tend to change it. Much editing in BioEdit requires extensive repetitive actions, so using the menus will rather slow. To change settings first create a new alignment File, New Alignment or open an existing file.
Next go View, Customize Menu Shortcuts. Select the value you wish to change, hit the value on the keyboard and that will reset it. On the lower toolbar 3rd of the alignment window, select the first solidly colored button.
This changes the way the sequences are displayed. On the middle toolbar 2nd in the alignment window change mode to edit, change box next to it to insert. Go View, save options as default. Close BioEdit, reopen your files and the settings should all be saved.