Workflows on designing, testing and storing primers in MacVector

By Chris | Published: June 4, 2018

MacVector is the best PCR Primer design application on macOS. It has many innovative primer tools to make designing, analyzing and cataloging your primers or oligos easy. Here are a few typical workflows.

Designing primers

Amplifying a gene

You can design a set of primers to amplify a gene in as little as three mouse clicks.

  1. Open your sequence.
  2. Open the MAP view, and click on a feature.
  3. Go to ANALYZE > PRIMER DESIGN/TEST(PAIRS).
  4. Click OK.

You will get a ranked list of the best primer pairs to amplify that feature along with a spreadsheet table, that you can copy and send to your oligo synthesis service.

Design a single targeted primer with a tail.

QuickTest Primer tool gives great flexibility for designing primers with tails or mismatches.You can slide your primer along your template to find the optimal sequence. You can add mismatches and view their affect on any CDS features. You can add restriction sites too and view “one out” sites.

  1. Select a 20 bp region around the location you want your primer to be.
  2. Click ANALYZE > QUICKTEST PRIMER.
  3. Slide the primer along your template until the oligo is optimal.
  4. Add a restriction site or mutation in the optional tail.
  5. Hover over a green (or red) putative restriction site to view the change to your sequence.
  6. Double click on that putative site to add to your primer. Watch how the coding is changed.
  7. Click Add to Database.. to save your primer.
MacVector

Testing Primers

How do I test a pair of primers?.

You can map pairs primers against your template to see how well they would amplify your target.

  1. Open your template sequence
  2. Go to ANALYZE | PRIMER DESIGN/TEST(PAIRS).
  3. For the left primer click USE THIS Primer
  4. Type or paste in your forward primer
  5. Repeat for the reverse primer
  6. Click TEST

Storing primers in the Primer Database.

MacVector’s Primer Database allows you to save and retrieve regularly used primers. You can also scan sequences for potential primer binding sites using Primer Database Search. The tool comes with a starter database of primers, but you can use existing subsequence files or import primers from Excel.

To save a primer from QT Primer to the Primer Database.

  1. Open ANALYZE > QUICKTEST PRIMER (INDIVIDUAL)
  2. Design your primer
  3. Click ADD TO…
  4. Give the primer a name and add a comment. Click OK

To save a primer from Primer Design to the Primer Database.

  1. Open ANALYZE > PRIMER DESIGN/TEST (PAIRS)
  2. Design your primer pair
  3. In the spreadsheet right click on a primer
  4. Choose ADD PRIMER TO DATABASE
  5. Give the primer a name and add a comment. Click OK

To use the Primer Database Search:

  1. Open your sequence
  2. Select ANALYZE > PRIMER DATABASE SEARCH
  3. Choose parameters and click OK

Design a primer to match an existing primer from the primer database

The Primer database allows you to store your own collection of primers. You can design new primers to match regularly used ones.

  1. Open your template sequence
  2. Switch to the Map tab and select the region you want to amplify.
  3. Go to ANALYZE | PRIMER DESIGN/TEST(PAIRS).
  4. For the left primer click USE THIS Primer
  5. Use the drop down menu to enter the existing primer from the primer database
  6. Click OK
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MacVector 16 and macOS High Sierra 10.13.4.

By Chris | Published: April 23, 2018

Apple have just released macOS® High Sierra 10.13.4. This may appear to be just an incremental update to macOS High Sierra. However, under the hood there are some major changes, not least is a new warning dialog if you are running an older application on macOS High Sierra.

With the release of macOS High Sierra 10.13.4 Apple have added warnings to all 32 bit applications and it is possible that with the next macOS release that 32 bit applications will no longer work. Apple have stated:

"....macOS High Sierra would be the last version of macOS to run 32-bit apps without compromise."

We’re pleased to inform you that MacVector 16 is fully supported and compatible with macOS High Sierra 10.13.4

However, older versions of MacVector are not compatible with this OS release and you may see the following dialog when you start MacVector 13.5 or earlier.

Blank Skitch Document

Here at MacVector we always strive to stay ahead of the game. When Apple recommended that all applications should be 64 bit, we immediately started the move to making MacVector a fully 64 bit application, culminating in the release of MacVector 14 in 2015. MacVector is a modern Mac application that takes full advantage of all the new technologies and ease of use of today’s macOS operating system.

If you are using an older version of MacVector then see what you are missing. Why not download a trial version, and request an upgrade quote.

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Assemble bacterial genomes in minutes on your Mac laptop

By Chris | Published: April 19, 2018

MacVector with Assembler contains some remarkably powerful algorithms for assembling Next Generation Sequencing (NGS) data. Not so long ago, you needed a powerful Linux server with lots of memory for de novo assembly of whole genomes. But with advances in the efficiency of algorithms and improvements in hardware, it is now possible to assemble quite large genomes on a Mac laptop.

MacVector 16 incorporates two separate NGS de novo assemblers, Velvet and SPAdes. Both are very capable assemblers with a small memory footprint. Velvet is significantly the faster of the two, but SPAdes often generates longer contigs as it does a slightly better job at resolving repeats, plus it can handle many more data types for mixed read assemblies and has a smaller memory footprint, allowing it to be used for larger data sets. With Velvet you often need to tweak the parameters for optimal performance, whereas SPAdes usually “just works”. SPAdes can often generate meaningful assemblies from relatively poor data where Velvet will fail without considerable tweaking of the parameters.

Both are invoked the same way: use File | New | Assembly Project to create a new project, then click on the Add Reads button and select the read files you want to import. Typically these are paired-end reads (either interleaved or as separate files), but they can be unpaired reads, consensus sequences exported from a different assembly, Ion Torrent, PacBio or Oxford Nanopore reads. You can also import compressed (gzip) files directly, with no need to uncompress them, saving a lot of disk space. Finally, click on the Velvet or SPAdes toolbar button to run the algorithms. The end result will be a number of contigs.

Here are some examples of performance, with all tests run on a 2013 2.7 GHz MacBook Pro with 16 GB RAM

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In the case of the small Mycobacterium genome, Velvet completed the assembly in a little over a minute. Even a moderately large ~7 Mbp Streptomyces sp assembly of 5 million HiSeq reads took just 16 minutes with Velvet and less than an hour with the more memory efficient SPAdes algorithm.

For a more in depth discussion of these results, please see our recent blog post.

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Simple Assembly of Sanger Sequencing Files with MacVector Assembler

By Chris | Published: April 19, 2018

With MacVector Assembler, assembling ABI Sanger Sequencing files is simple, fast and accurate. MacVector uses the popular phred/phrap/cross_match set of tools from the University of Washington. To improve accuracy, and to help resolve repeats, these tools use “quality scores” (popularly known as “phred scores”), giving them an advantage over many other methods. To assemble two or more ABI files, follow these steps.

  • Use File | New | Assembly Project to create a new project
  • Click on the Add Seqs toolbar button and select all of your ABI (or SCF) chromatogram files to import
  • Click on the phred toolbar button – this re-calls the traces and generates quality scores (no need to select any items in the project, though you can to run phred on specific files)
  • Click on the phrap toolbar button and accept the defaults

    • After phrap has run, you will be presented with one or more contigs (assuming your ABI reads actually overlap). If you double-click on one of those, a contig editor will open letting you view and edit the actual alignments.

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    Simple DNA sequence assembly on a Mac with MacVector with Assembler.

    MacVector has a software plugin called Assembler that integrates directly into the DNA sequence analysis toolkit and provides DNA sequence assembly functionality. Dealing with sequencing reads has never been easier.

    MacVector includes no less than five different assemblers just a few mouse clicks away from your sequencing reads. Phrap assembles Sanger sequencing reads or existing contigs, while there are three separate NGS de novo assemblers – Velvet for short read datasets, Flye for Nanopore and PacBio long reads and SPAdes for mixed assemblies. For reference assembly Bowtie2 can map millions of sequencing reads against genomic reference sequences and is ideal for RNASeq gene expression analysis data too.

    Assembler is tightly integrated into MacVector. It’s easy to bring sequencing reads into MacVector, and it’s just as easy to directly design primers for a contig, run BLAST searches on a contig, and much more, right from your desktop!

    Not sure if you have Assembler? Choose MacVector | About MacVector. If the screen that appears says “MacVector with Assembler, Pro Edition” then you have it. If not, you can sign up for a fully functional 21 day trial version.

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    Assembling sequencing data with MacVector and Assembler

    By Chris | Published: April 19, 2018

    MacVector has a software plugin called Assembler that integrates directly into the DNA sequence analysis toolkit and provides DNA sequence assembly functionality. Dealing with sequencing reads has never been easier.

    MacVector includes no less than five different assemblers just a few mouse clicks away from your sequencing reads. Phrap assembles Sanger sequencing reads or existing contigs, while there are three separate NGS de novo assemblers – Velvet for short read datasets, Flye for Nanopore and PacBio long reads and SPAdes for mixed assemblies. For reference assembly Bowtie2 can map millions of sequencing reads against genomic reference sequences and is ideal for RNASeq gene expression analysis data too.

    Assembler is tightly integrated into MacVector. It’s easy to bring sequencing reads into MacVector, and it’s just as easy to directly design primers for a contig, run BLAST searches on a contig, and much more, right from your desktop!

    To assemble various types of sequencing reads, follow these steps.

    NewImage

      • Choose File | New | Assembly Project to create a new empty project file.

    Then follow one of the following:

    To create a de novo assembly from Sanger reads

      • Click on the Add Reads tool bar button, then select the sequence files you wish to assemble and click on the Open button. Read(s) file(s) can also be drag and dropped on the open Assembly Project window.
      • Click Phred to basecall sequences in the project. Note that if no sequences are selected, phred will be run on ALL of the files in the project.
      • Click Phrap to assemble the reads.

    To create a de novo assembly from NGS datasets

      • Click on the Add Reads tool bar button, then select the sequence files you wish to assemble and click on the Open button. File(s) can also be drag and dropped on the open Assembly Project window. Paired reads files are automatically detected.
      • Choose either SPAdes or Velvet to assemble the reads.

    To create a de novo assembly from Nanopore or Pacbio datasets

    • Click on the Add Reads tool bar button, then select the sequence files you wish to assemble and click on the Open button. File(s) can also be drag and dropped on the open Assembly Project window. Paired reads files are automatically detected.
    • Double click on the reads and ensure they are correctly flagged as Pacbio reads.
    • Choose Flye to assemble the reads.

    To create a reference assembly

    • Click the Add Reads button, then select the sequence files you wish to assemble and click on the Open button.
    • Click Add Ref, select the sequence file(s) you wish to align the reads against and click on the Open button.
    • Click Bowtie to map all read files against all of the reference sequences in the project.

    Not sure if you have Assembler? Choose MacVector | About MacVector. If the screen that appears says MacVector with Assembler, Pro Edition then you have it. If not, you can sign up for a fully functional 21 day trial version

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    MacVector video tutorials on You Tube

    By Chris | Published: April 19, 2018

    There are short video tips on using MacVector on our blog and our YouTube channel. Each one is less than 2 minutes and generally shorter than that! There will be a new screencast every few weeks, so please subscribe to our YouTube channel so you don’t miss any!

    The latest screencasts include quickly annotating a gene to a sequence, confirming a small sequencing project against a reference and checking the orientation of a ligated insert using MacVector’s Restriction Digest and Agarose Gel tools.

    If there’s any tools or features you’d like to see a screencast about please do let us know…. and always remember we will come and run real live workshops for you too!

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    Viewing external database entries for features in a sequence.

    By Chris | Published: April 19, 2018

    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

    By Chris | Published: April 19, 2018

    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

    By Chris | Published: April 19, 2018

    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

    By Chris | Published: April 19, 2018

    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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