Sequence Assembly: What can Assembler do for my lab?

By Chris | Published: March 13, 2023

Assembler is fully integrated into MacVector and allows you to manage sequencing data with the familiar MacVector ease.

de novo sequence assembly using Phrap, Velvet and SPAdes with Flye for PacBio and Oxford Nanopore.

Reference Sequence Assembly: Map millions of reads against genomes, transcriptomes or other reference sequences using Bowtie2.

MacVector

Compare Genomes: Compare two related annotated genomes to see common or missing genes.

Coverage Tab: compare multiple assemblies with expression level comparison.

Variant Calling: SNPs and INDELS are visualised on your assembly and supplied in VCF.

Bacterial genome tools Tools for finishing bacterial genomes including circularizing genomes.

Easy to use interface. Navigating around your assemblies has never been easier. Display an entire contig in the graphical Map and select a read to zoom straight to that region. Click on a base in a contig and see coverage and variants.

MacVector

Heterozygote analysis. Analyse heterozygotes in Sanger trace files and assemblies.

Assembly Project manager makes it easy to assemble multiple datasets, reference sequences and assemblies. Work with unaligned reads from an assembly directly in the project.


Compare different assemblies. Map multiple datasets against the same reference.

RNA-Seq analysis with read depth visualization and per gene coverage data (RPKM & TPM).

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Setting the Numbering Origin

By Chris | Published: March 2, 2023

Preserving sequence numbering is particularly useful if you want to work on a smaller more manageable region of a large chromosome but wish to retain the original numbering. When you copy a section of a larger sequence and paste the copy into a new MacVector sequence window (or use FILE | NEW FROM CLIPBOARD), the original numbering is retained.

SettingOrigin

  • Open pBR322 (in the SAMPLE FILES folder in the MacVector application folder).
  • Either:
    • in the EDITOR tab select the Features popup menu and select the tetracycline resistance CDS.

      • or

    • click on the TET feature in the MAP tab.

    This selects the region from 86 to 1276 in both the EDITOR and MAP tabs.

  • Now choose Edit | Copy, followed by File | New from Clipboard. A new window appears with the numbering origin set to 86. (You can also accomplish this by choosing File | New | Nucleic Acid and then Edit | Paste into the new window).
  • If you want to quickly reset the origin to “1”, you can right-click (or -click ) in the sequence area to bring up a context sensitive menu and choose Reset Origin to 1.

    • Changing the numbering of existing sequences

    You can also easily change the numbering of a sequence by selecting then dragging the small red cross that usually appears at the beginning of the sequence.
    Dragging the cross to another location designates that as the “plus 1” residue – all residues before that position will be given negative numbers.
    You can also set the first residue to a positive number. To set this, double-click on the red cross and enter a new start value in the sheet that appears.

    Screenshot 2023 03 01 at 14 00 53

    Setting the Circular Origin

    If you are working with a circular sequences then you can change the location where the sequence is “split” in the editor.

    you can just right click the sequence and set SET CIRCULAR ORIGIN from the context sensitive menu that will appear. This also changes the Map tab so that the new position is located at 12 o’clock.

    In the MAP tab you can also select a restriction enzyme site and right- click to choose Set Circular Origin.

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    MacVectorTip: Selecting the sequence from a single restriction enzyme site to the end of a linear sequence

    By Chris | Published: February 16, 2023

    To see the distance between any two points on a sequence is easy. For example select one restriction enzyme site, hold down SHIFT and select the second. The start, stop and length will be shown in the Range Selector (top right corner of every window – see images below). But if you want to see the distance from a Restriction Enzyme site to the end (or beginning) of the sequence it is slightly more difficult.

    You have two options.

    • Select the RE site in the MAP tab.

    • Switch to the EDITOR tab.

    • Hold down SHIFT then click right at the end of the sequence. You can also hold down SHIFT, press the RIGHT cursor key, then the DOWN cursor key.

    RE Selection EDITOR

    You can also do this entirely within the MAP tab. Although this is slightly more complicated as you need to change the type of cursor.

    • In the Graphics Palette select the SELECT SEQUENCE cursor button

    • Now you can drag and select sequence in the MAP tab, exactly as you can do in the EDITOR tab with the other cursor.

    RESelection MAP
    Both of these options will select from the restriction enzyme site right to the end of the sequence and show the length in the top right corner.

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    MacVectorTip: How to Customize the Toolbars of MacVector windows

    By Chris | Published: February 9, 2023

    Like many Mac applications, MacVector takes full advantage of macOS’s ability to add, delete and rearrange the action buttons on window toolbars. To make these changes, right-click (or [ctrl]-click) in the gray space on any toolbar and a context-sensitive menu will appear. Choose Customize Toolbar and a dialog will be displayed with all of the buttons available for that tab, like this one for the Editor tab of the DNA Sequence Window.

    EditingToolbar

    Note that modifying the toolbar is a global change that affects all windows containing that tab. It is also specific to different document types, so you can have different sets of buttons on the Editor toolbar of the DNA, Protein, Trace/Chromatogram and MSA document windows for example. Once modified, the changes remain permanently until you either customize them again, or reset your MacVector Preferences.

    You can also customise the Analyses Toolbar with your most often used tools. This will be a global change and remain the same for all windows.

    CustomizeAnalysesToolbar

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    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.

    Unknown

    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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