How to call heterozygotes in trace files or Assembly Projects

By Chris | Published: January 19, 2023

In our latest release, MacVector 18.5, we added a new tool to call heterozygotes in sequencing reads.

The heterozygote analysis tool allows you to either view heterozygotes in Sanger trace files or to permanently change the basecalled sequence with an ambiguity representing the called heterozygote. The tool works on multiple trace files in the Assembly project manager or the Align to Reference editor. You can also run it on a single trace file in the Single Trace Editor

HET analysis Trace

How to run Heterozygote Analysis

To view putative heterozygotes

Select Trace files in the Align to Reference editor or Assembly Project Editor or open a trace file in the the single trace sequence editor.
  1. Run ANALYZE – HETEROZYGOTE ANALYSIS
  2. An Options dialog will appear. Change the options and click OK
  3. A summary dialog will appear showing the number of heterozygotes found across how many sequences.
  4. Click OK
  5. A new tab will appear in the Results window showing the location of the possible heterozygotes.
You can click on the highlighted blue position to be taken to the heterozygote. Note if you are in Assembly Projects or Align to Reference editors then the heterozygote will be displayed in the Single Trace Editor.

To permanently basecall the sequence

Assembler

  1. Add trace files to your Assembly Project
  2. Select one more more trace files
  3. Select ANALYZE | BASECALL | USING HETEROZYGOTE ANALYSIS
  4. An Options dialog will appear. Change the options and click OK
  5. A summary dialog will appear showing the number of heterozygotes found across how many sequences
  6. Click OK

Align to Reference

  1. go to FILE | NEW | ALIGN SEQUENCES TO A REFERENCE
  2. Choose your reference sequence
  3. Add trace files to your Assembly Project
  4. Select one more more trace files
  5. Select the BASECALL toolbar button or ANALYZE | BASECALL | USING HETEROZYGOTE ANALYSIS
  6. An Options dialog will appear. Change the options and click OK
  7. A summary dialog will appear showing the number of heterozygotes found across how many sequences
  8. Click OK

Trace File Editor

  1. Double click to open a trace file.
  2. Select ANALYZE | BASECALL | USING HETEROZYGOTE ANALYSIS
  3. An Options dialog will appear. Change the options and click OK
  4. A summary dialog will appear showing the number of heterozygotes found.
  5. Click OK
Once run there is a new BASECALL line showing the new sequence. In the Assembler Project editor an H will appear in the status column. Heterozygotes are indicated by an ambiguity.
Ensure the BASECALL toolbar button is toggled to green to view basecalled lines.

Settings

The default settings should be enough for the majority of trace files. However, there are a number of settings that can be adjusted where needed. The default value is in brackets.
  • Normalise peak heights using a window of (25) residues
  • Percent of each peak width to use (50%)
  • Minimum number of normalised residues (3)
  • Minimum Heterozygote threshold (35%)
  • Minimum Base Call threshold (75%)
  • Ignore low quality regions (yes)
  • Where a window of (21) residues
  • Has an average quality value of less than (20)

HET analysis Assembly

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MacVectorTip: Changing the font used in the Editor, Results and other windows

By Chris | Published: January 19, 2023

EditorFontsPrefs

MacVector is very customizable in how you can produce graphical maps of sequences, assemblies, alignments and more. You can also change the default appearance of MacVector itself. The font used in the Editor and Results window can be changed and increased in size. You are limited to using fixed width fonts (such as Andale Mono anc Courier) as otherwise sequences will not align properly. But you can change the font size. Additonally You can change the size of the font used in the Graphics Palette. All very useful for very large monitors to avoid squinting at the screen!

  • Open MacVector | Settings…
  • Choose Font.
  • Change the settings and close the Settings dialog.
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    MacVectorTip: macOS Dark Mode and forcing MacVector to open in Light Mode

    By Chris | Published: January 12, 2023

    “Dark Mode makes it easier to stay focused on your work because your content stands out while darkened controls and windows recede into the background”

    If you use Dark Mode with the Auto setting, then with the short winter days in the northern hemisphere (for our southern hemisphere friends please save this email for six months!) you will be spending a lot more time in Dark mode than those long summer days.

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    Using Dark Mode is a very different user experience and may not be to every user’s taste. For example as can be seen in the screenshot above plasmid maps with dark backgrounds can be a little “odd” at first. So if you like working with Dark Mode, but would prefer MacVector to use Light Mode then read on.

    You have always been able to change this with a command line to change the preferences. However, in the last minor release (MacVector 18.5.1) we exposed this setting in the MacVector Preferences so you do not need to use Terminal.

    To force MacVector to always open in Light Mode

    This new setting is in MacVector | Settings | General | Always use Aqua

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    You will need to restart MacVector.

    If you are using an older version then you can still do this from the command line.

  • close MacVector
  • type or copy/paste the following command into the Terminal window
    • defaults write com.macvector.MacVector NSRequiresAquaSystemAppearance -bool YES
  • When you start MacVector again, it should be running in Light Mode.
  • If you want to reset that preference, open Terminal and type/paste this command:
    • defaults delete com.macvector.MacVector NSRequiresAquaSystemAppearance

    To support Dark Mode we made a considerable number of design changes to MacVector’s user interface, so that toolbar buttons and Map tab colors suit both Dark and Light modes. Nonetheless MacVector’s default colors were originally designed with Light Mode in mind so the colors may not always be ideal for your needs. Do remember that MacVector gives you a lot of control over the default appearance of the display. See the following preferences pane: MacVector | Preferences | Color

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    Merry Christmas from all of us at MacVector….!

    By Chris | Published: December 24, 2022

    We hope 2022 was a productive and enjoyable year for your research, and that MacVector has played some part in that. Once again, 2022 was not the uneventful year that we all hoped for. Perhaps 2023…?

    Before you go and relax with friends and family, help your colleagues make the most of MacVector and give the gift of MacVector Weekly Tips for Christmas. We take these tips from the most common support calls and emails that we receive. Please do encourage any of your colleagues who use MacVector to sign up.

    It only takes a few seconds, but the gift lasts all year. They will only receive MacVector emails once a week and we never pass on email addresses to third parties.

    We wish you a happy, healthy and rewarding New Year! Here’s to a wonderful 2023 and thank you for using MacVector.

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    MacVector 18.5.1

    By Chris | Published: December 21, 2022

    We’ve just released a minor update, MacVector 18.5.1.

    Changes include:

  • Further enhancements for importing Sequencher projects (.spf) including heavily edited assemblies.
  • A new setting for always opening MacVector in “light” mode even when macOS switches to “dark” mode.
  • Easier remote activation of standard licenses for larger sites.
  • increased number of seats for serverless network licenses.

    • You will be automatically promoted to update. or go to FILE | CHECK FOR UPDATES… to update now.

    You can download the full installer instead.

    For assistance with remove activation of standard licenses please contact support.

    Screenshot 2022 12 21 at 13 47 40

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    MacVector 18.5 has just been released and is macOS Ventura ready!

    By Chris | Published: November 11, 2022

    It’s that time of year again. Apple have released macOS Ventura and we are very pleased to announce MacVector 18.5 is out. MacVector 18.5 is fully macOS Ventura ready!

    MacVector running on macOS Ventura

      macOS Ventura ready

    • MacVector 18.5 was developed and tested on macOS Ventura. It is supported on macOS High Sierra to macOS Ventura.
    • MacVector 18.5 is a Universal Binary application and will run natively on Apple Silicon M1 and M2 Macs as well as Intel Macs.

      • Heterozygote Analysis of Sanger trace files

      Heterozygous analysis of an assembly

    • The heterozygote analysis tool analyzes one or multiple Sanger trace files and reports on all possible heterozygotes.
    • You can also analyze Sanger trace files and permanently change the basecalled sequence with an IUPAC ambiguity code representing the basecalled heterozygote.
    • The tool works on multiple trace files in the Assembly project manager or the Align to Reference editor.
    • You can also basecall heterozygotes in a trace file in the Single Trace Editor.

      • Heterozygous analysis of a single trace file

      Align to Reference supports long reads

    • Long sequencing reads from PacBio and ONT sequencers can now be assembled in Align to Reference. 

      • Miscellaneous enhancements

    • Importing Sequencher project (.SPF) files has been significantly enhanced.
    • As usual there’s many bug fixes and changes that you probably don’t care too much about! But be assured that all we do keeps MacVector future proof and to be a modern macOS application that you can rely on.

    How to upgrade to MacVector 18.5

    If you have a maintenance contract that was active on 1st November, 2022, then you can install MacVector 18.5. You must be running macOS High Sierra to macOS Ventura. You will be prompted to automatically update within the next few days.
    You can also download the installer and do it manually now.

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

    Even if you have downloaded the trial in the past then downloading a new trial will give you a fresh 21 days to evaluate MacVector.

    When a trial license expires it becomes MacVector Free. So if you decide against upgrading then you can just delete the trial license and easily go back to your current version. It’s risk free as MacVector files are backwards compatible.

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    MacVector 18.5 is macOS Ventura ready

    By Chris | Published: October 25, 2022

    It’s that time of year again. Apple have just released macOS Ventura and we are very pleased to announce our current release, MacVector 18.2 has been tested on macOS Ventura and is compatible.

    Not only that but we have just a few weeks of testing left before our next release. MacVector 18.5 has been developed on macOS Ventura since the first developers beta build was available. MacVector 18.5 has been extensively tested on macOS Ventura too.

    MacOSVentura desktop 1200

    Compatibility of previous versions

    For versions of MacVector before the current one you can check compatibility on a table which we update after every official release of macOS.

    macOS compatibility of older versions Table

    • For versions of MacVector released over the past few years it is likely that they will work fine. Our developers strive to future proof MacVector, and it is only when Apple make significant changes that older versions may stop working.

    • Such significant changes include:
      • MacVector 13.5 and all older versions will not run on macOS Catalina and earlier. This is due to Apple moving fully to a 64 bit operating system.
      • MacVector 14.0 relies on an Apple library that Apple removed in MacOS Big Sur and later macOS releases.
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    MacVector is macOS Ventura ready

    By Chris | Published: October 25, 2022

    It’s that time of year again. Apple have just released macOS Ventura and we are very pleased to announce our current release, MacVector 18.2 is macOS Ventura ready.

    Not only that but we have just a few weeks of testing left before our next release. MacVector 18.5 has been developed on macOS Ventura since the first developers beta build was available. MacVector 18.5 has been extensively tested on macOS Ventura too.

    MacOSVentura desktop 1200

    Compatibility of previous versions

    For versions of MacVector before the current one you can check compatibility on a table which we update after every official release of macOS.

    macOS compatibility of older versions Table

    • For versions of MacVector released over the past few years it is likely that they will work fine. Our developers strive to future proof MacVector, and it is only when Apple make significant changes that older versions may stop working.

    • Such significant changes include:
      • MacVector 13.5 and all older versions will not run on macOS Catalina and earlier. This is due to Apple moving fully to a 64 bit operating system.
      • MacVector 14.0 relies on an Apple library that Apple removed in MacOS Big Sur and later macOS releases.
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    MacVectorTip: Create custom Codon Usage Tables for ORF analysis and reverse translation

    By Chris | Published: October 19, 2022

    MacVector uses codon usage tables (also called “Codon Bias”) to help you predict protein coding regions in the Analyze | Nucleic Acid Analysis Toolbox functions and also the protein Analyze | Reverse Translation... function. For example, here are the Plus and Minus strand Staden Codon Preference plots aligned under the Plus/Minus ORFs for a Streptomyces coelicolor cosmid fragment using the Streptomyces coelicolor.bias codon usage file.

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    While MacVector includes a collection of common codon usage tables (Human, Mouse, Drosophila, E. coli etc.) in the /MacVector/Codon Bias Tables/ folder, there may be times when you need a custom table. We do have a utility that can convert data in the appropriate format into a MacVector .bias file, but unfortunately it will not yet run on recent macOS releases. However, we are happy to generate these files for you if you send us the raw data. To do this, go to http://www.kazusa.or.jp/codon/ and search for your preferred organism. You should ask to see the data in the CodonFrequency GCG format.

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    Copy the text starting from AmAcid to the end of the data.

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    Paste it into an email and send it to support@macvector.com and we will send you back a .bias file.

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    macOS’s tabbed windows and MacVector

    By Chris | Published: September 23, 2022

    One of the lesser known features of macOS is the ability to store all open documents of an application in tabs. Tabs were initially introduced for the Finder, but macOS Mavericks saw them apply to supported application document windows too. MacVector has supported tabs since their introduction, however, by default the Tab Bar is turned off.
    To view the Tab Bar in MacVector then use:

    VIEW | SHOW/HIDE TAB BAR

    UnknownHowever, to control the behaviour when you open new documents you need to use the main system preferences dialog:

    SYSTEM PREFERENCES | GENERAL | PREFER TABS [NEVER | IN FULL SCREEN | ALWAYS] WHEN OPENING DOCUMENTS.

    When set to ALWAYS then every time you open a new document in all supported applications, then it will open in a new tab. If you prefer multiple windows you can drag the tab out of the window to open in a new window. However, you may prefer for some windows to be tabbed and others to always open in separate windows. So if you want a particular sequence in a separate windows, then drag the tab out of the window or WINDOW | MOVE TAB TO NEW WINDOW

    Please note that MacVector’s Results windows are always tabbed irrespective of the SYSTEM setting. However, you can always drag a results tab out of a window to open in a new window.

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