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!

How to check the orientation of a ligated insert using MacVector’s Restriction Digest and Agarose Gel tools.

How to check the orientation of a ligated insert using MacVector’s Restriction Digest and Agarose Gel tools.

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Annotating a gene in MacVector

Creating biologically accurate AND beautiful maps of your plasmids is easy in MacVector. All you need to do is select some sequence, then open the FEATURES EDITOR. Every annotation is fully compatible with the Genbank specification and Feature Table.

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Downloading hits from the MacVector 15.5 BLAST Map results tab

The MacVector 15.5 BLAST Map results tab is a unique interface for examining the annotations around hits to a query sequence. Each pane in the display represents a High Scoring Segment Pair, as seen in the BLAST Aligned Sequence tab. At the lower left corner of each pane is a Download button – when you click on it, a popup menu appears with several options.

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The major options are to either download the entire genome, remembering this might be many Mbp in size, or to just download the displayed fragment. In general, MacVector displays the High Scoring Segment Pair region of the sequence, plus 2kb on either side to provide context, and that is what will be downloaded. To reduce the number of mouse-clicks when scrolling through a lot of results, Download to Last Folder places the downloaded sequence in the last folder that you selected (or your Downloads folder if you’ve never selected one before).

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Controlling Automatic ORF Display

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

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

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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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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 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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Generating a primer report to send to your Oligo Synthesis service

QuickTest Primer is a great tool for primer design. Paired with Primer Design/Test (Pairs) it gives you great control and flexibility for designing primers with tails, mismatches, silent mutations, one out sites and more.

Once you’ve designed your primer the next step is to get it synthesized. QuickTest Primer will produce a PDF report of your primer with a single click on the Report button. The report contains all the information you need about your primer, including sequence, any mismatches between the primer and template, any restriction sites you have created, the melting temperature of the oligo, molecular mass, %GC and much more. Great for sticking in your lab book or emailing to the oligo synthesis service.

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How to toggle between 1 and 3 letter amino acid codes

MacVector displays amino acid translations in many different result windows. You can drill down to the residue level in the Map tab and see translations of CDS and other translatable features and see translations in the plain text views and the Quicktest Primer interface. The translations can be viewed as either single letter codes or as triple letter codes. This is controlled by the Preferences | Text View pane. Simply toggle this and click on the Apply button and all of the open views will update with the new setting.

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MacVector’s compatibility with different versions of macOS/OS X

We strive to ensure MacVector has good forwards and backwards compatibility. For example, MacVector 15.1 will still open files created with the very first version of MacVector! However, since Apple releases a new operating system every year, and generally make fairly significant “under the hood” changes, it is just not possible to support every version of OS X and macOS. Although we strive to ensure new releases of MacVector are compatible with as yet unreleased versions of OS X/macOS, Apple do not always make future changes public and so it is not always possible!

The table below shows which OS releases are officially supported on each version of MacVector. Please contact the Support team if you have any questions. See our website for more details.

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Clone construction using Digest/Ligate rather than Copy/Paste

We’ve previously looked at using Edit | Copy and Edit | Paste to quickly and simply create new constructs using Restriction Enzyme sites. Here’s 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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Confirming a small sequencing project against a reference sequence

Align to reference is a perfect tool for mapping small sequencing projects against a reference sequence. It’s perfect for accurately and quickly:
– Confirming the sequence of a cloned fragment
– Sequencing across the ends of a cloned fragment to confirm the junction sequence
– Screening clones from a site-specific mutagenesis experiment to identify successful mutations
– Screening related clones for single nucleotide polymorphisms

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