General musings from the MacVector team about sequence analysis, molecular biology, the Mac in general and of course your favorite sequence analysis app for the Mac!

Use the BLAST Map to better identify blast hits

With the advent of cheap Next Generation Sequencing technologies, there has been an explosion of whole genome sequences deposited in BLAST databases. One consequence of this is that, particularly for sequences of bacterial origin, most of the significant hits are to entire genomes. The classic BLAST results show the sequence alignments, but give no indication of what features are present on the database sequence at the alignment location. MacVector 15.5 has a new unique BLAST Map result tab that shows the actual annotations around the alignment location for each high scoring segment pair.

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If a sequence is highly annotated, such that not all of the features are visible, simply holding the mouse over the appropriate pane will expand the pane to display all of the features. If you have already annotated (or partially annotated) your query sequence, those features will appear in the grey background “overlay” pane – that stays locked to the top of the window while the hits can be scrolled underneath it.

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Controlling The Automatic ORF Display in MacVector 15.5

MacVector 15.5 automatically scans every DNA sequence window for open reading frames and displays the results in the Map tab.

AutoORFExample

The setting for this are controlled by the MacVector | Preferences -> DNA Map pane, along with the automatic Show restriction sites settings.

DNA Map Prefs

The Minimum Number of Codons setting is fairly obvious. 5’ ends are starts and 3’ ends are stops are used for linear sequences and will display ORFs coming into the sequence that do not have a start codon, or ORFs that read out of the sequence without a stop codon. Codons after stops are starts is useful if you just want to know the locations of the longest possible ORFs in a sequence, irrespective of the existence of a start codon. Finally, the Suppress annotated CDS ORFs check box prevents the display of ORFs that are already annotated. However, if a potentially longer ORF is present, e.g. there is another ATG codon upstream, that ORF will be displayed. You can read more details here.

The appearance of the ORFs can be controlled using the Options | Default Symbols menu item.

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MacVector 15.5 is out: Graphical BLAST and automatic ORF display.

Our latest release, MacVector 15.5, introduces an entirely new way to view the results of BLAST searches, with an interactive graphical interface that let’s you easily visualize where your query maps to a hit. Unannotated Open Reading Frames are now automatically displayed whenever you open a DNA sequence. A number of changes to the Align To Reference and Contig Editors simplify the task of closing gaps and resolving repeats in sequencing assemblies.

BLAST MapResults

Graphical BLAST Results

Displays a fully annotated alignments of your query sequence against each high scoring hit. Genes and other features are displayed for both query and hits. This allows you to clearly see which genes your query is mapped to. For each hit, you can also just download the alignment only. Very useful when your query is a single gene, but hits are genomes or chromosomes.

ORF AutoAnnotate

Automatic ORF Display

MacVector now automatically searches for open reading frames in every DNA sequence that you open. You can quickly annotate the ORF to your sequence with a right mouse click.

Align to Reference

Many new tools have been added for closing gaps, and resolving repeats, in sequencing assemblies.

Misc changes

A variety of menu changes will make many tools more accessible to less experienced users. A number of optimizations have been made to better handle opening an analyzing extremely large sequences and reference alignments.

How to update to MacVector 15.5

If you have active maintenance and are running MacVector 13.0.1 or later then you should have been notified about the new release already. At that point you have the option to automatically upgrade to MacVector 15.5. To install this version, you must have a maintenance contract that was active on 1st February, 2017. You must also be running OS X 10.7 or later.

If you have an older version of MacVector then download the trial and request an upgrade quote.

If have downloaded the trial in the past then downloading a new trial will give you a fresh 21 days to evaluate MacVector even if a previous trial license had expired.

Remember that when a trial expires it becomes MacVector Free.

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Clone construction using Digest/Ligate rather than Copy/Paste

In previous post we looked at using Edit | Copy and Edit | Paste to quickly and simply create new constructs using Restriction Enzyme sites. This post will look at an alternative approach, using the Digest and Ligate buttons and the Cloning Clipboard.

The sequence window Map tab has two buttons called Digest and Ligate. These work in a very similar manner to Copy and Paste. If you select a restriction fragment, Digest becomes active.

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The difference between Digest and Copy is that instead of copying the fragment to the global system pasteboard, Digest copies it to MacVector’s internal Cloning Clipboard.

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The Cloning Clipboard keeps a history of every fragment you have Digest’ed, though it’s easy to remove individual fragments or reset the entire clipboard.

The Ligate button is very similar to invoking Paste, except that instead of pasting the contents of the global system clipboard, it pastes the selected fragment on the Cloning Clipboard. Again, if the ends are not compatible, a dialog will be displayed letting you fill or cut back the ends to force a blunt ended ligation.

You can also join fragments together on the Cloning Clipboard by simply clicking in the circles at the end of fragments, then dragging and dropping those onto a different end.

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Simple but accurate restriction enzyme based clone construction using Copy and Paste

The quickest and simplest way to create restriction enzyme generated constructs in MacVector is to use Edit | Copy and Edit | Paste. The strategy to use is identical to copying a paragraph from one Microsoft Word document to insert into a second document. i.e.

  • Select the restriction enzymes flanking the source fragment in either the Map tab of the sequence window, or the Map Results tab after a Restriction Enzyme analysis. Hold down the [SHIFT] key to select the second enzyme. In circular maps, the selection always proceeds in a clockwise direction, so the order you select is important.
  • Choose Edit | Copy. This places the fragment onto the system clipboard, along with sticky end information.
  • Switch to the destination vector. Select the target restriction enzyme site(s). When selecting two sites, make sure you select the fragment you want to replace.
  • Choose Edit | Paste. If the restriction enzyme site(s) have ends compatible with the copied fragment, this will insert the fragment from the system clipboard into the vector. If the ends are incompatible, you will get a warning “ligation dialog” with options to fill or cut back ends to make them compatible.
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    The red line around the left ends indicates that they are compatible. If you were to fill or cut back the right hand ends you would see green lines indicating they are now compatible.

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    MacVector will never let you create an invalid construct, so you can select/copy/paste to create new constructs safe in the knowledge that if the ends are not compatible, MacVector will always show this warning dialog.

    One final tip, if a fragment can be inserted in either orientation, hold down the [option] key and the Edit | Paste menu item will change to Edit | Paste Flipped, letting you insert the fragment in the opposite orientation.

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    How to change the default appearance of features

    If you download a sequence from Entrez, or open a sequence that is not in MacVector format, MacVector assigns a default appearance to any features on the sequence. For example, CDS features are shown as a blue arrow with a Helvetica 9 point font label. With the advent of ultra high resolution displays, you may find that the default font is a little too small to easily view. MacVector has a Default Symbols dialog that lets you control the appearance of every default symbol. To invoke this, hold down the [option] key and choose the Options | Default Symbols… menu item. Note that if you have just started MacVector and have no open sequence windows, or if a non-sequence window is front-most, you do not need to hold down the [option] key.

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    To change the font used for CDS features, select the Features tab, then click on the DNA CDS item in the list of feature types. Then you can click on the font button to select a preferred font using the system-wide font selection window. You can change the font for multiple features types at once by selecting multiple rows in the list using the or keys then selecting an appropriate font.

    Note that with the current version of MacVector (15.1) you will need to close and then re-open sequence files for the change to take effect.

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    Use File->Export in the Contig Editor to save selected reads

    Last week we covered the fact that you can use File->Export to save sequences or alignments in different formats. Delving down deeper into this, some of the views will save different types of data based on what you select in the format menu. The best example of this is the Contig Editor. If you choose File->Export… in this window you get the following File Format options;

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  • MacVector Reference Assembly – this saves a copy of the alignment in MacVector format. It is the same as choosing File->Save As….
  • MacVector NA Sequence – this saves the Reference sequence only as a MacVector formatted .nucl file. If you have edited the Reference, this is the easy way to save a copy of it under a new filename.
  • FASTA Multiple Sequence – this saves any selected reads into a single fasta-formatted multiple sequence file. Use this if you have aligned a large number of sequences and want to save a subset of them into a single file for additional analysis (e.g. assembly).
  • FASTQ Multiple Sequence – similar to the fasta option, but saves in fastq format. This format includes quality information.
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    Use File->Export to save sequences in a different file format

    Over the years, Apple have changed the recommended layout and functionality of the File menu. At MacVector, we always try to follow Apple’s guidelines wherever possible so that new users will feel comfortable with familiar menus. However, long-term users may experience some confusion, particularly if you want to save sequences in a different format. Here is the definitive guide to the Save options in MacVector.NewImage

  • Save – this saves the current sequence (or alignment) in MacVector format.
  • Save As… – this saves the current sequence (or alignment) under a different name, but always in MacVector format. The open sequence/alignment window will change its name so that any subsequent Saves will act on the new file.
  • Export… – this is the menu option to use if you want to save a copy of the sequence/alignment in a different format e.g. GenBank, EMBL, fasta, fastq etc. There is a file format option displayed in the resulting dialog that lets you choose the format of the file. As implied in the menu name, this does not change the name or file location of the sequence/alignment.
  • Export Tab Contents As… – All of the above menu options act on the entire document. This menu option applies only to the data displayed in the current tab. So, in the example above, which is a graphical view of the sequence, selecting this will give you the option of saving the image in a variety of formats (PDF, tiff, png etc). Tabs that contain text will give options to save in different text formats, whereas “spreadsheet” type views give the option to save in tab-delimited or comma delimited formats.
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    How to reset the graphics palette if it disappears

    The floating graphics palette is extremely useful for helping to configure the display of the Map tab. You can easily toggle the visibility of any individual or group of features and adjust many aspects of the display. MacVector does try to remember the last position you moved this to so its there ready for you when you restart MacVector. Sometimes, especially if you plug into external monitors then close your computer and open it up without shutting down MacVector, that location may be off-screen. While MacVector does try to compensate for this, there are occasions where the Graphics palette stubbornly remains offscreen. If this happens to you, the easiest way to restore the graphics palette is to download and run this simple utility.

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    When you run it, make sure you click on the “Yes” button!

    The utility will attempt to close and re-open MacVector after you click “Yes”. You will be prompted if there are any unsaved documents. Generally, it is better to run this after you have quit MacVector.

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    Tips for finding ORFs in your sequence

    We’ve talked in previous tips about annotating open reading frames as CDS features. However, what if your sequence has no annotated ORF? MacVector’s ANALYZE | OPEN READING FRAMES… tool will help you find any quickly.

    However, if you are new to this tool there are a few options that may prove initially confusing. These options modify how ORFs are detected. They are intended to help you find ORFs when you may only have a partial fragment of a gene. So if you’ve just ran an ORF analysis and find too many CDS regions or if you cannot find an ORF and you are not confident you have the full gene, then remember this email!

  • 3’ ENDS ARE STOPS means that in any sequence it looks at each reading frame and checks whether the last full CODON would give a ORF, that is longer than the minimum number of codons. Put simply it assumes that the end of the sequence is the end of a coding region, even if no STOP codons are present.
  • 5’ ENDS ARE STARTS does the reverse. That is assumes the start of the sequence is a START codon.
  • CODONS AFTER STOPS ARE STARTS assumes that after every STOP codon there is a new START codon. Even if the actual codon is not present.
  • In the two screenshots below we have a sequence with a single long CDS region (in blue). If we have these two options turned on then you will see three ORFS on the three forward frames (top image). If you turn them off you see a single CDS (bottom image).

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    Don’t forget to annotate your CDS region when you’ve found it. Many tools in MacVector will show additional information about a coding region if it is annotated as a CDS region. If you simply drag the ORF from the RESULTS window it will automatically annotated that as a CDS region. It will also include the coding region’s translation directly in the annotation.

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