Organising your sequences using Smart Folders

By Chris | Published: July 4, 2016

OS X’s Finder has many features for quickly finding and working with your files. Spotlight Search is one such tool that most Mac users are familiar with. However, Smart Folders is a tool that is very useful but often overlooked.

Smart Folders allow you to create a dynamic folder whose contents are derived from a Spotlight Search. The folders are permanent but dynamically updated, and can even be shown directly in the sidebar of Finder.

You can have very simple Smart Folders or complex ones. Smart Folders are very useful for keeping track of your MacVector sequences. For example “show me all Assembly Projects last modified within the past month” or “Show me all trace files from the past seven days“.

To create a Smart folder to show all MacVector sequences last opened in the past month:

  • Click FILE | NEW SMART FOLDER in Finder.
  • Click “+” next to SAVE
  • Enter KIND is OTHER “macvector” for the search terms
  • Click “+”
  • Enter LAST OPENED DATE is WITHIN LAST – 1 – MONTHS
  • Click SAVE and enter a name. Don’t forget to include it in the Sidebar.

    • Check out the Smart Folders blog post for more information.

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    How to increase the number of graphics levels to stop features overlapping.

    By Chris | Published: July 4, 2016

    MacVector tries to optimize the Map graphics layout using a trade-off between performance and minimizing unnecessary white space. Sometimes the default settings we have chosen are not ideal, particularly if you are looking at the Map tab of an Align To Reference window where you have a large number of reads overlapping the same region. To clean up these views, you can increase the number of graphical levels using the MacVector Preferences | Map View tab.

    For more information on the use of levels in the Map tab, please checkout this blog post.

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    Know Your Alignments

    By Chris | Published: June 13, 2016

    We often get asked “how do I do an alignment” using MacVector? Well, the answer to that is always “it depends”, and it depends on what you want to learn about your sequence(s). Here’s a quick summary of the different types of alignments and what you would use them for:

  • Multiple Sequence Alignment (File | New | Protein Alignment or Analyze | Align Multiple Sequences Using | …) Use this when you want to explore the relationships between sequences. This is primarily aimed at protein sequences. Unless you want to explore the phylogenetic relationship between nucleic acid sequences (e.g. rRNA) there are almost always better approaches to use for DNA sequences.
  • Align To Reference (Analyze | Align To Reference). If you are working with DNA sequences, this should be the first function you consider for alignments. Use this to align one or more nucleic acid sequences against a reference. It handles regular sequences, ABI chromatogram files and even NGS data. This is the ideal interface for resequencing and mutagenesis identification experiments. The algorithm is strand aware enough to automatically “flip” sequences as appropriate and can even align cDNA sequences to a genome, accounting for introns and splice sites.
  • Dot Plots (Analyze | Create Dot Plot | …). This is a great and underrated function for quickly comparing 2 sequences to get an overview of the similarity between them. You can use this to quickly check that two sequences do have some similarity, but you can also use it to compare two genomes and see were the identities, duplications and inversions are located. This is also a good way to see the extent of local alignments between two sequences.
  • BLAST. (Database | Internet BLAST Search). MacVector’s implementation of the standard NCBI BLAST search. But still a great way of getting a quick idea of which sequences your sample is related to.
  • Align To Folder. (Database | Align To Folder). Like BLAST, but for your own personal sequences. Handles fasta and fastq files so that you can use this to pick out reads from a huge database that match your sample sequence.
  • Primer Database. (Analyze | Primer Database Search). A relatively new addition to MacVector, but you can use this to align a collection of primers to a test sequence. This lets you define tails for your primers and you can even select a pair of primers in the graphical result window and Edit | Copy will copy the predicted amplification product, complete with any engineered mismatches and overhanging tails.

    • You can learn more about the different alignment options in these three blog posts, Alignments in MacVector, Aligning Trace files, and Aligning primers to a Reference sequence.

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    Importing sequences/features from websites such as ENSEMBL or UCSC’s Genome Browser

    By Chris | Published: June 13, 2016

    Many Genome Browsers/databases allow you to browse and view genomes, or a specific gene/region, with a high degree of detail. For example ENSEMBL and the UCSC Genome Browser. However, many times you want to work with that data on your own Mac. As usual the easiest way to exchange sequence data is using the Genbank format. This stores ALL biological annotation about a sequence and is of course fully supported by MacVector.

    However, a better way is to “refresh” your own sequence with the latest current data, using MacVector’s IMPORT FEATURES tool. This will use annotation, using a format such as GFT, GFF or BED, from a genome browser and import to an existing sequence. Very useful for keeping your own sequence, current with known and published annotation about that sequence.

    The following example uses ENSEMBL, but the general workflow applies to other Genome Browsers:

  • Choose the region of interest
  • Click EXPORT DATA

    • To import a Genbank file:

  • in OUTPUT choose GENBANK and click NEXT
  • Choose the TEXT format.
  • SELECT ALL | COPY
  • Switch to MacVector and use FILE |NEW FROM CLIPBOARD.

    • To import annotation using IMPORT FEATURES:

  • in OUTPUT choose GFF3 (or BED/GFT) and click NEXT
  • Choose the TEXT format.
  • Save the file.
  • Switch to MacVector and use FILE | IMPORT FEATURES.

    • Read more about importing annotation in general and importing from ENSEMBL.

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    How to reset the zoom level when you Drag Zoom into your sequence.

    By Chris | Published: June 13, 2016

    Being able to Drag Zoom makes it easy to view a specific region of your sequence in greater detail. To Drag Zoom, just hold down the mouse button and drag the cursor along your sequence. The Map view will redraw to show the selected area in greater detail.

    Once you are zoomed in you have a few options to choose the region, or to zoom back out.

  • A simple double click on the background of the Map tab will reset the zoom to show the full sequence map.
  • You can also use the RESET ZOOM button in the Graphics Palette to do the same.
  • The ZOOM IN and ZOOM OUT buttons in the Graphics Palette will allow you to fine tune the level of zoom.

    • The cursor keys also work:

  • Left and right will slide around a circular sequence, or along a linear sequence.
  • Up and Down will vary the level of zoom.

    • As well as using Drag Zoom you can also zoom magnify the entire sequence. Use the magnification cursor in the Graphics Palette to do that. Then hold down the OPTION key to reverse that when you click.

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    Generating a primer report to send to your Oligo Synthesis service

    By Chris | Published: June 13, 2016

    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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    Selecting sequence residues in the Map tab

    By Chris | Published: June 13, 2016

    The Map tab is very flexible and allows you to do almost all of the sequence manipulations you might need, without switching tabs. In addition to selecting segments of a sequence by clicking on Restriction Enzyme sites or Features, you can also directly select sequence even when the residues are not visible and represented by the sequence line. Prior to MacVector 14, you had to click on the Sequence Selection mode button in the floating graphics palette to achieve this.

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    Now you can select the sequence directly even in the default “zoom” mode. Just carefully position the cursor over the sequence, click and drag to select the region you are interested in. This is easiest to do when the Map tab is zoomed to the residue level, but with care you can do it at all zoom levels.

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    The main advantage of this functionality is that you never really need to switch to the Editor tab unless you actually need to type residues. Between selecting Features, Restriction Sites or the sequence itself, you can prepare the sequence for manipulation by copying or pasting, or selecting a region to apply an analysis or create a new feature.

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    ASM Microbe 2016

    By Chris | Published: June 10, 2016

    The MacVector team will be in Boston next week (16-20 June) for ASM Microbe 2016.

    We’re looking forward to visiting Boston again. It’s a great city.

    We’re on booth 441. Please do drop by and say hello. We’ll be able to show you the upcoming release of MacVector 15 and MacVector for Windows. There’ll probably be some candies too!

    The hashtag for the meeting is #asmmicrobe2016.

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    Testing pairs of primers

    By Chris | Published: June 6, 2016

    Quite a few versions back MacVector used to have an old, but well loved, Test PCR Primer Pairs algorithm. Its interface was clunky and it did not take into account modern algorithms for calculating Tm. However, the results were useful. If you liked this tool then you will be pleased to know that the functionality is still in MacVector.

    When you enter a pair of primers into Primer Design/Test (pairs) (previously called Design Primers) the interface switches to a new testing mode, TEST PRIMER PAIRS. This tool lets you test pairs of primers using an updated variant of the Test PCR Primer Pairs algorithm.

  • Open ANALYSE > PRIMER DESIGN/TEST (PAIRS)
  • Switch both fields to USE THIS PRIMER and type the primer sequences you want to test into the two edit boxes.
  • The tool switches to TEST PRIMER PAIRS
  • Click on the OK button to run the test.
  • A Summary window will appear showing problems in red text.
  • Select the Test Output, Spreadsheet and Graphical Map checkboxes and click OK.

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    Creating a custom set of restriction enzymes containing just the enzymes in your freezer drawer.

    By Chris | Published: June 6, 2016

    MacVector has multiple tools for displaying restriction enzymes sites in your sequence. All of these tools use Restriction Enzyme files. These are a set of files, updated regularly from the REBASE database, grouped according to reagent supplier. Whereas the default file is “Common Enzymes” if you only purchase enzymes from NEB, then you can choose the NewEnglandBiolabs.renz file each analysis.

    It is very easy to create a custom set of enzymes. If you stock a regular set of enzymes in your lab’s freezer then you can easily create your own restriction enzyme file with just your freezer drawer’s contents! When you open a sequence and see a site you know you have that enzyme available without having to order it.

    How to create your own custom RE file

  • create a new empty file; FILE > NEW > RESTRICTION ENZYME.
  • Save your new file in a safe location.
  • Open the Commercial Restriction Enzymes.renz file (this contains all enzymes).
  • Now drag and drop sites from this file to your new one.
  • To search, type the enzyme name in the top right control (change the filter to NAME).
  • Once you have finished don’t forget to save it.
  • Click PREFS in the toolbar, switch to the MAP View tab and click SET ENZYME FILE.
  • Select your newly created file and click CHOOSE.

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