MacVectorTip: Quality scoring of manual edits to your contigs.

Quality scoring of Assemblies and Align to Reference alignments can be visualized directly on the sequence. Residues can be shaded according to their quality scores. These can be displayed anywhere quality values are available, including de novo and reference assemblies in Assembler and Align to Reference alignments.

The intensity of the shading of residues indicates the phred-based quality value of each residue.

  • For individual reads, this ranges from 0 (deep red) through 20 (white) to 40 or above (deep green). The consensus scale is doubled and ranges from 0 (deep red) through 40 (white) to 80 or above (deep green).
  • Gaps are always shown with a white background. You can “mouse-over” a residue to view the numerical information in a tooltip.
  • Edited residues are always given a phred quality value of 99 and these residues are given a blue background.

Most assembly algorithms are quality score aware and better quality reads will take priority over lower quality sequences. However, edited residues will always override all other sequences/reads even if they are high quality. This also means that a single read with an edited sequence will take priority over many other reads with a different sequence and the edited residues in that single edited read will define the consensus. This is done as we assume that the user knows best!

Here five edited residues override all other sequences and are shown in the consensus.
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How to design a digest to screen minipreps after a ligation.

MacVector’s Agarose Gel tool can be used to quickly design a restriction digest to screen minipreps following a ligation. 

(View full size on website…)

Replicate your ligation in MacVector.

  • Select the two sites, for subcloning your targeted gene, and click DIGEST.
  • Drag the digested fragment from the Cloning Clipboard to your vector
  • click LIGATE.

Create your agarose gel with the correct insert and a vector only lane.

  • Open your sequence and switch to the MAP tab.
  • Drag a restriction site (or sites) that digest within the fragment and also in the vector to the Agarose Gel window.
  • Open up your original cloning vector.
  • Drag the same site(s) to the Agarose Gel window.

Undo the ligation, and repeat with the wrong orientation.

  • Switch back to the ligated sequence, and use UNDO to remove the ligated fragment.
  • Switch back to the Cloning Clipboard.
  • Drag the same digested fragment from the Cloning Clipboard to your vector.
  • Hold down [OPTION] and click LIGATE.
  • Switch to the MAP tab.
  • Drag the same site (or sites) that digest the fragment and the vector to the Agarose Gel window.

Now you will end up with an Agarose Gel with three lanes: A lane with empty vector, a lane with the insert in the correct orientation, and a lane with the insert in the wrong orientation. Now it’s easy to screen your minipreps, as you know the gel bands of a correct miniprep before you’ve even loaded it on the gel!

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MacVectorTip: Grayed out graphics indicate Missing Features

If the graphics in a nucleic acid sequence Map tab appear somewhat “washed out” it is because the graphic items represent common features that MacVector has found that are not annotated on the sequence. For example, here are the Map and Feature tabs of an unannotated cloning vector;

You can see a number of features on the Map tab, but the Features tab is completely empty. The graphics indicate common features that MacVector has identified that have not been annotated on the sequence. If you select one of the features in the Map tab and right-click (or [ctrl]-click) there is an option in the resulting context sensitive menu to Add CDS Feature. When that is selected, the feature takes on a bold appearance and a new annotation appears in the Features tab.

If you wish, you can select multiple missing features and then add them all with a right-click. Or you can select the Results | Missing Features tree view item in the floating Graphics Palette to select all missing features and then add with a right-click.

Note that the automatic display of missing features is controlled by the MacVector | Settings | Scan DNA tab. From there you can control how they are identified or even point the algorithm to your own folder of annotated sequences to be a source for the missing features.

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MacVectorTip: “Nudge” Reads for Better Reference Alignments

The MacVector alignment algorithms are usually pretty good at finding the optimum alignments of reads against a reference sequence. But, very occasionally, they may get confused by repeats or other anomalies in the sequences. Or perhaps you have made after-the-alignment edits: for example, in the Align to Reference Editor, you can insert residues by holding down the option key while typing a residue rather than the normal overwriting editor. For example holding down option and pressing delete will delete a residue rather than replace it with a gap. In such cases, you may want to “nudge” the read left or right to maintain a better all around alignment without resorting to repeating the alignment algorithm. You can do this by selecting the name of the read you want to “nudge” then pressing the left or right arrow keys to move the entire sequence relative to the reference.

Here is a misaligned read before pressing the [right] arrow.
And after pressing the [right] arrow
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MacVectorTip: Trimming by Quality in sequence assemblies

Many of our users may be familiar with the ability of Sequencher to semi-automatically trim poor quality sequences from the ends of Sanger ABI reads. Although it is generally not necessary to do this in MacVector because most of the algorithms can automatically handle poor quality data, there are times when it can be beneficial. MacVector has a Quality Trimming function that removes residues from the ends of Sanger reads that fall below a configurable quality threshold. You can invoke this in either the Align to Reference or Assembly Project windows by clicking on a new Qual Trim toolbar button.

This opens a setup dialog letting you determine how the reads should be trimmed.

The trimmed residues are normally shown greyed out.

But can be completely hidden by clicking on the Trimmed toggle toolbar button.

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MacVectorTip: Use self comparison matrix analysis to identify repeats and inversions

The Dot-Plot analysis (Pustell DNA Matrix) function in MacVector is an extremely powerful way of quickly getting an overview of the similarities between a pair of sequences. However, it can also be used to identify repeats and inversions in a single DNA sequence simply by comparing a sequence to itself. For example here is the Alcohol Dehydrogenase gene cluster from Drosophila funebris.

There is a direct tandem duplication of one set of genes which can clearly be seen by the presence of additional lines that are not on the main “identity” diagonal.
You can use this to identify inverted repeats as well. The display is interactive so that you can zoom in to any part of the plot by a simple mouse drag to go from this;

To this:

The image above shows the inverted terminal repeat from a Bovine herpesvirus with the inverted nature of the repeat indicated by the blue colored lines that go from bottom left to upper right.
More complicated structures can often be seen.

In this example there is a tandem direct duplication where each repeat itself consist of 7 direct overlapping repeats.
You can also use Dot Plots as sanity checks when running de novo sequence assemblies. Here is an assembly of what should have been a 6.5kb circular plasmid that the assembly algorithm assembled into a 28kb linear sequence consisting of 4 direct copies of the plasmid. This is not uncommon with very noisy long read NGS data where algorithms might assume the high error rate is actually a series of SNPs;

You can also view the textual alignments in the Aligned Sequence tab. That data also updates when you zoom in to a specific region.
Hint: If you try this yourself and get a lot of background “noise”, try increasing the Min. % Score parameter from the default 60% to 80% or higher.

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MacVectorTip: Restriction enzyme sites and tooltips

Quickly viewing the recognition sequence and cut site of a restriction site is very easy in the Map tab.

By default MacVector’s Scan DNA For… tool will automatically display restriction enzyme recognition sites in the Map tab. If you hover your mouse over a restriction site, a tooltip will show you the restriction enzyme recognition site, the location of the cut site, and number of times that enzyme cuts your sequence.

– Make sure Preferences | Scan DNA | Restriction Sites is turned on:

You can also see the full sequence and cut site when zoomed to sequence level in the Map tab.

– In the Graphics Palette click the Zoom to Residue button.

Unique sites are shown in red, whereas enzymes that cut at two or more sites are shown in blue.
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MacVectorTip: Advanced Align to Reference Editing

You can use the Analyze | Align to Reference function to align other sequences (Sanger chromatograms, plain sequences or even NGS data collections) against a reference. Once aligned, the Editor lets you perform all the usual editing functions using an “overwrite” mode – select the residue you want and type the new residue to replace it. Typing a “space” or a gap “-” will delete the residue. However, there are a number of other editing functions you should be aware of:

  • Hold down the [option] key and type a residue to insert a residue, rather than overwrite. The new residue inserts before the selected base.
  • Hold down [option] and type [delete] to delete a residue rather than replace it with a gap. This will cause the residues to the right of the deletion to slide to the left by one character.
  • To “nudge” an entire read, simply select the “name” of the read in the top left pane and press the [left] or [right] arrow keys.
Here is a misaligned read before pressing the [right] arrow:
And after pressing the [right] arrow:
  • There are a large number of editing functions that can be accessed through a context-sensitive right-click menu. MacVector 18 introduced a new Extra “hamburger” menu that shows the same list:
  • Most of these operate on the entire set of selected read(s) and the actions are fairly self-explanatory. However, some may need additional explanation:
    • (i) Select Matching Pairs – primarily relevant for NGS data, this will attempt to select the corresponding mate for paired-end reads, based entirely on the name.
    • (ii) Select Overlapping Reads Containing Selected Sequence – if you select a short sequence in one of the reads, perhaps containing a SNP, this will select all of the sequences that contain that same SNP.
    • (iii) Close Gaps by Deleting Residues – with large alignments, you may be distracted by a column of mostly gaps due to e.g. one read out of 100 having an additional inserted residue. This function removes these occasional insertions to clean up the alignment.
    • (iv) Extend Reference with Selected Read – only works if a single read is selected that extends past either end of the reference sequence. Useful for building up “patch” sequences when trying to resolve repeats during genome assemblies.
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MacVectorTip: How to Toggle Between Single-Letter and Three-Letter Amino Acid Translation Code

Many views in MacVector display amino acid translations above or below DNA sequences. Typically, these are from CDS features, but can also be the 3/6 frame translation of a sequence. You can display the amino acids as either the single-letter code, or as the three-letter code. You can toggle the setting using the MacVector | Preferences -> Text View pane.

Changes are reflected in many different windows. Here are a few examples.

Single Sequence Editor tab
Single Sequence Map tab when zoomed to the residue level.
Quicktest Primer window
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MacVectorTip: Drag and drop in the Gibson Assembly window

Over the years, we have added a lot of drag and drop functionality to MacVector. Of course, as with any application, it is not always obvious that you can drag and drop to accomplish tasks because you literally have to drag and drop to discover you can do it at all! So here is the first of an occasional series pointing out some of the functionality in MacVector where drag and drop can save you time.

Gibson Assembly: MacVector supports designing primers for Gibson Assembly cloning projects along with other primer-based ligase-independent approaches such as the popular “In-Fusion” system. You can create a new project via the File | New | Gibson/Ligase-Independent Assembly… menu item. Then you need to add a “vector backbone” to get started. Often, this might simply be a restriction digested vector that you throw into the mix – if so, open the vector, select the chosen restriction site and drag it into the window.

You can then open other annotated sequences and drag and drop features that you would like to include in your final construct. 

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