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!

Viewing external database entries for features in a sequence.

Sequences, or regions of sequences, can be linked to external databases. For example an entire sequence entry or for when annotation tools are used to annotate proteins with domain or motif information (for example InterProScan). Very useful for when you want to view more detailed or updated information. Within the Genbank specification, which MacVector extensively uses, an external database entry can be stored in a /DB_XREF qualifier. This allows the database entry to be easily viewed. The Genbank (and Genpept) specification allow for many different databases to be accessed using this qualifier.

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In MacVector the original database entry can easily be viewed in a web browser by selecting, then right clicking the feature entry in the Features tab and viewing the available DB_XREF entries. Selecting one will load it in your web browser.

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Use the Replica Button For Synchronized Views

Most primary MacVector windows (Nucleic Acid Sequence, Protein Sequence, Multiple Sequence Alignment, Align To Reference, Contig Assembly etc.) have a Replica toolbar button. If you click that button, a second window will open, potentially set to a different tab. The key to this functionality is that the two windows are linked – any selections you make in one window will reflect in the other. In most cases this means that if you select an object in, for example, the Map tab of one window, the Editor tab of the other window will actually scroll to display the selected region.

Here’s an example where clicking on an aligned read in an Align To Reference Map tab has automatically scrolled the replica Editor tab to show the selected sequence.

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How to Identify Bacterial Promoters Using MacVector

MacVector’s Subsequence tool is a very flexible search function that can be used for a variety of tasks. MacVector itself has a built-in variant of the function for maintaining and search primer databases (Analyze | Primer Database Search…). Each entry in the file MacVector uses as a source of subsequence data can have up to 3 segments, with variable length between the segments, along with a defined number of permitted mismatches and even a system for requiring that specific residues must match. That makes it ideal for searching for bacterial promoters. For example, the canonical Escherichia coli promoter sequence is a “-35” region TTGACA, then a gap of 16 to 18 residues, then a “-10” region “TATAAT”. You will find there is an EcoliPromoter.nsub file in the /MacVector/Subsequences/ folder. If not, you can download it. If you open the file in MacVector, you can see this.

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You can see that the file has four entries – each of these has two segments representing the -35 and -10 region, but each has additional settings that control how close a match has to be before it is reported. The names give some idea of the stringency of the match – Perfect, Probable, Possible and Weak. If you double-click on the Probable item, you get this editor.

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Import Multi-Sequence Genbank Files into an Assembly Project for easy access to Features

There are many genomes in the Genbank database that cannot be downloaded as single annotated sequences. These might be large multi-chromosome eukaryotic genomes, but, increasingly, partially sequenced bacterial chromosomes where the major contigs have been annotated using the NCBI annotation pipeline. Typically, when you encounter these, there are options to download annotated versions of these as multi-sequence Genbank formatted files. MacVector has the option to open any file containing multiple sequences as either a Multiple Sequence Alignment document or as individual Sequence documents. This is not always optimal if you have more than a handful of sequences in the file. However, if you use MacVector with Assembler, you can import these sequences into a project using the Add Ref toolbar button and the individual sequences will not only be displayed in the project window, but, if you double-click on one, the complete annotated sequence will be opened.GenbankintoAssemblyProject

This is a great way to view and/or sort collections of annotated sequences in Genbank format that cannot be done directly through the Apple Finder. Once opened, you can Export… any sequence in another format if you wish.

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Opening multiple sequences as alignments or individual sequences

Many sequence formats contain multiple concatenated sequence entries. For example FASTA and Genbank are two formats capable of storing multiple individual sequences.

By default MacVector will treat such sequences as alignments and open them in the Multiple Sequence Alignment editor. Most users who want to open such a file do want to see an alignment. Additionally if the default behaviour was to open as individual sequences, then accidentally clicking on a large alignment would result in many hundreds of individual sequence windows opening up on your desktop (do remember that holding down the OPTION key and clicking on the close button will close all open sequences).

If you need to open such a sequence file as individual sequences, then there’s a simple option that you need to check in the FILE | OPEN dialog. This behaviour has not changed for quite some time. However, back in MacVector 13 the appearance of the dialog changed, due to a change in Apple’s current guidelines on file dialogs. Whereas the older dialog had an obvious way to see this dropdown menu, now all you see is a small OPTIONS button in the bottom left hand corner.

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To open multiple sequence files as individual files you need to check an option in the FILE | OPEN dialog.

  • Click FILE | OPEN
  • In the dialog click OPTIONS (bottom left corner)
  • Change OPEN MULTIPLE SEQUENCE FILE AS from AUTO to SINGLE SEQUENCES
  • Click OPEN
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    Restoring file associations when MacVector no longer opens your sequences

    Macs are pretty good at choosing the right application to open a document. For example when you double click on a .nucl document then it will open in MacVector. However, sometimes this file association breaks. Applications should coexist peacefully on a Mac, but sometimes a misbehaving app will corrupt these file associations and you will find that your sequence displays a generic document icon (or what’s worse a different application!). When you double click on the icon, it will no longer open in your favourite DNA sequence analysis tool!

    Luckily this is easily fixable:

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    1. Select a .nucl file in the Finder
    2. Choose File | Get Info (or use command-I).
    3. In the “Open with” section, click on the popup menu and select MacVector
    4. Then click on the Change All… button to apply the change to all files.
    5. Repeat for all file types used by MacVector that are not opening correctly (e.g. .prot, .msan, .msap, .axml)
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    What can MacVector do for me?

    Here’s what MacVector can do for your lab.

    Comparing sequences

    Whatever type of alignment your sequence needs, there’s a tool in MacVector.

    Cloning

    CRISPR Indel Analysis: Identify insertions and deletions following CRISPR editing of a target.

    Sequence assembly of NGS data against a reference genome or compare your sequencing against your new construct.

    Translated Multiple Sequence Alignments: Align DNA sequences based on their translations.

    Align proteins against a reference great for comparing known proteins against an unknown one.

    Auto Annotation of common plasmid features to blank sequences.

    InterProScan: Scan proteins for functional domains against many databases.

    Cloning

    MacVector

    Design Cloning workflows

    As simple as dragging a fragment to a cloning vector.

    Flexible Cloning Subclone with restriction enzymes, Gibson cloning, Gateway and more.

    Cloning history Every step is documented.

    Agarose Gel to run out digested sequences. Easily identify site(s) to differentiate successful clones.

    Primer Design

    Design primers with ease.

    QuickTest Primer changes primer design. Hairpin? Nudge your primer until it goes.

    Add tails to your primers with silent restriction sites/mismatches and view reading frame changes.

    Quickly design pairs of primers click a region to get the best primer pairs to amplify it.

    Primer Database store your primers and scans sequences for potential binding sites.

    MacVector

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    Searching and downloading sequences from Entrez

    MacVector has integrated connectivity to the NCBI BLAST and Entrez databases. You can directly search Entrez for DNA or Protein sequences based on features, authors, keywords etc and directly download them into MacVector, complete with all features and annotations.

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    Eastern Great Lakes workshop tour in February

    The MacVector team will be touring the Eastern Great Lakes for a series of workshops in February.

    We will be running workshops in Rochester NY, Buffalo NY, Ypsilanti MI, Cleveland OH, Wooster OH, Columbus OH and Cincinnati OH.

    Monday, Feb. 5th

  • 10:00 – 12:00 University of Rochester, Rochester, NY
  • 2:00 – 4:00 University at Buffalo, Buffalo, NY
  • Tuesday, Feb 6th

  • 10:00 – 12:00 Wayne State University in Detroit, MI
  • 2:30 – 4:30 Eastern Michigan University, Ypsilanti, MI
  • Wednesday, Feb 7th

  • 10:00 – 12:00 Cleveland Clinic Foundation, Cleveland, OH
  • 2:00 – 4:00 College of Wooster, Wooster, OH
  • Thurs. Feb. 8th

  • Nationwide Children’s Hospital in Columbus, OH.
  • Friday, Feb 9th

  • 11:00 – 1:00 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
  • In workshops we try to highlight the new functionality introduced over the last few years to MacVector that even more experienced users may not be familiar with. The format is very informal and participants are encouraged to ask questions and help direct the workshop towards areas of the most interest. We’ve found that every workshop we have run has helped users make the most of MacVector.

    (more details to follow).

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    Using MacVector’s Auto Annotate tool to annotate blank sequences.

     

    Have you ever been sent a plain unannotated sequence, or downloaded a sequence from Entrez and been disappointed as it doesn’t have the carefully curated graphical appearance of your favorite genes? Auto annotation solves both of these common problems. The basic idea is that you can scan the sequence against a folder containing a collection of existing annotated sequences and MacVector will find the matching features in the folder and add those to the starting sequence.

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