How to identify methylation blocked Restriction Sites

By Chris | Published: February 19, 2016

MacVector currently does not have a built-in function for recognizing restriction sites that are blocked by the DAM or DCM methylases. However, there is a simple workaround that lets you visually identify those sites in the restriction enzyme analysis result windows. The basic idea is that you create additional restriction enzyme sites for each enzyme containing the recognition sequence that is affected by methylation. E.g. ClaI (ATCGAT) will not cleave the sequence ATCGATC. So we can create a DAM-ClaI recognition sequence in a MacVector formatted restriction enzyme file

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When you search a sequence with this file, you can immediately see which ClaI sites will be blocked by methylation;

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Too tedious to type in all those methylated sites? Not to worry, we’ve done all the hard work for you! You can download a pre-formatted file and copy/paste the full set of DAM-blocked enzymes into your own favorite file. If you are interested in learning more about this, you should definitely check out the complete blog post.

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Downloading BLAST Hits from the NCBI

By Chris | Published: February 18, 2016

When you use MacVector to run a BLAST search, did you know that you can download any matching sequences directly from the BLAST Description List window? Simply select text on any part of the line(s) representing the sequence you want to download and choose Database | Retrieve To Desktop or Database | Retrieve To Disk to download the selected sequences.

Retrieve To Desktop will download and open each selected sequence in a window on your desktop. Retrieve To Disk will prompt you for a location on your hard drive – the better option if you want to download more than just a few sequences.

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Creating cloning construction flowcharts in third party applications

By Chris | Published: January 28, 2016

We’ve previously discussed how every ligation is documented. You get a “Frag” annotation that contains the date, source sequence, enzymes used and any end modification that was done to that fragment during the ligation.

However, we had regular requests to make it easier for user to document their constructs in other ways. For example being able to construct a flow diagram showing how a construct was made for your lab book.

So in MacVector 14.5 we added PDF export to the Cloning Clipboard. Now you can copy and paste a fragment as a PDF in a single step. The image can be pasted into any graphical application, such as Illustrator.

This makes it easy to create a graphical illustration of a cloning strategy. The fragment is copied, exactly as it is displayed in the Cloning Clipboard showing restriction enzymes that have been used to digest it.

  • Select the fragment in the Cloning Clipboard.
  • Use EDIT > COPY
  • Move to your application and use EDIT > PASTE

    • All fragments are copied as linear images, so if you need to show a circular vector, then in the Map view just copy and paste that.

    In this first example here’s a document (in Omnigraffle) showing a simple ligation.

    CloningClipboard

    The fragments are copied at a fixed width size, irrespective of the actual fragment sequence length. However, if you need finer control over your fragment before creating a PDF of it, then simply double click on the fragment. It will appear in a new MacVector sequence window which you can modify before copying it as a PDF.

    In the second document here’s three fragments all inserted into a single Gateway vector via Multisite Gateway cloning.

    CloningClipboard2

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    Clearing the “find” history in the Primer and Find dialogs.

    By Chris | Published: January 21, 2016

    Many tools in MacVector store a history of sequences, or search terms, that you have previously used. For example the Find dialog and Primer Design tools. This is accessed using a drop down menu to the right of the box where you would normally type, or paste, your sequence.

    This is to allow easy access to previously used sequences. Quite useful when you are searching with the same primer over many sequences in the same session (consider using the Primer Database for more permanent access to the same primer).

    However, sometimes you may want to wipe these and start afresh. Especially if you’ve mistyped a primer sequence, and keep forgetting to not use it!

    All you need to do is right click, or CTRL click on the dropdown menu and you’ll access a menu that allows you to remove an entry or clear the history entirely.

    In the primer tools don’t confuse this with the drop down menu to access the primer database.

    ComboBoxWithHistory and 14 5 nucl Editor

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    Simulated Agarose Gels

    By Chris | Published: January 18, 2016

    MacVector 14.5 has a Agarose Gel interface which allows you to view photo-realistic recreations of restriction digests of linear and circular DNA molecules. The gels look so realistic that users have had a hard time telling photos of their own digests from the simulation in MacVector. When you first use the new tool and compare it to your real gels, you’ll see what they mean!

    The new tool makes designing digests for checking constructs very easy and quick. For example, to check the orientation of a cloned gene, drag a restriction site in that gene to the gel window to view the correct band pattern. Then for comparison, repeat the ligation in the incorrect orientation and drag the site again. You can even print out the gel to take into the darkroom as a guide for cutting out a band.

    GelAnimationForTip
    It’s incredibly easy to use the new functionality:

    For a single digest

  • FILE > NEW > AGAROSE GEL
  • Open your sequence and switch to the MAP tab.
  • Drag a restriction site from the Map tab and drop on the Gel window

    • For a double digest

  • FILE > NEW > AGAROSE GEL
  • Open your sequence and switch to the MAP tab.
  • Select one restriction site, hold down SHIFT and select a second restriction site.
  • Drag the sites from the Map tab and drop on the Gel window.

    • To change the gel marker

  • Click the ADD MARKER toolbar button.
  • Choose a new DNA marker.

    • To remove a lane

  • Select the lane.
  • Drag and drop the lane outside of the gel window.
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    MacVector 14.5 is out!

    By Chris | Published: January 4, 2016

    We are pleased to announce that MacVector 14.5 was released at the end of 2015.

    To upgrade to this version if you are running OS X 10.7 or later then just go to MACVECTOR > CHECK FOR UPDATES… to allow MacVector to automatically upgrade. If you are running OS X 10.6 then you will need to download the installer.

    To install this version, you must have a maintenance contract that was active on November 1st 2015. You can check your maintenance expiry date by using MACVECTOR > ABOUT MACVECTOR... If you are not eligible for the upgrade then you can upgrade for a very reasonable price!

    There’s a lot to like about MacVector 14.5 but there’s one new tool that we are all very excited about, and we hope that you are too! MacVector 14.5 has a new photo-realistic Agarose Gel simulation window, that’s incredibly easy to use. Just create a new Agarose Gel window with File | New | Agarose Gel, then drag restriction enzyme sites from any sequence to your Agarose Gel window as illustrated in this animation:

    GelAnimationForTip

    If you want a double digest, then just drag two sites across.

    The migration of markers and digestions is very accurate and will make it easier to guess which band to cut out in the darkroom. You can specify % agarose, what markers to use, what dye fronts to display and much more.

    Do let us know what you think about this new tool.

    Another new feature is being able to copy fragments from the Cloning Clipboard as PDF to let you document cloning strategies in third party applications, such as Adobe Illustrator, or other graphical packages.

    MacVector 14.5 is packed with too many tweaks and enhancements to fully list here but it’s well worth pointing out its optimised memory footprint for working with large sequences and datasets. Align to Reference is especially faster when scanning large datasets of paired reads. Plus Align to Folder has had a lot of tweaks to help deal with large datasets of sequencing data and is now paired read aware.

    MacVector 14.5 is fully compatible with all 64 bit Macs running OS X 10.6 Snow Leopard to OS X 10.11 El Capitan.

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    Happy holidays from all of us at MacVector!

    By Chris | Published: December 24, 2015

    ChristmasWithSnow2015

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    Cloning Clipboard: A few tips on working with digested fragments

    By Chris | Published: December 16, 2015

    The Cloning Clipboard is an easy, and flexible, way to design and document your cloning strategies. Recently we discussed how every ligation in a cloning procedure is documented and stored in a sequence, so you always know how a construct was made.

    If you need to manipulate a fragment as a sequence, before ligating it, then it’s easy to do so. Just double click on a fragment in the Cloning Clipboard and you’ll create a new sequence window containing your fragment with its annotation. From now you can treat that as a normal MacVector sequence. Do remember though that the “digested end” information will be lost and in any ligation the new sequence will be treated as a blunt ended fragment.

    630 to 5530 of VectorA plus 649 to 2064 of VectorB plus 735 to 1483 of VectorC Map and Cloning Clipboard

    If you drag a fragment from the Cloning Clipboard to a vector, then you’ll get the ligate dialog to allow you to ligate it into that vector. However, if you have already selected a pair of enzyme sites, then the ligation dialog does not appear. Instead your fragment is immediately ligated into those two sites. This also happens if there is an unambiguous way the two fragments would ligate together. For example an EcoRI-BamHI fragment would ligate directly into a vector digested with EcoR I and BamH I. However, an EcoR I-EcoR I fragment would not ligate directly into a vector digested with just EcoRI.

    Screenshot 16 12 2015 17 26

    If you drag a fragment to the Desktop, then you’ll get the usual “Clipping” file containing only the sequence of your digested fragment.

    In the next release (MacVector 14.5 is out very, very soon!) you’ll be able to easily generate flow charts showing how a construct was made. The Cloning Clipboard will allow you to export fragments as PDF, rather than sequences!

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    101 things you (maybe) didn’t know about MacVector: #48 – Setting the Alignment match/mismatch characters

    By Kevin | Published: December 9, 2015

    There are many output windows throughout MacVector that display aligned sequences. If you run an Align To Folder, Create Dotplot or Internet Blast Search, one or more of the output windows will show alignments in a plain text format with some sort of character indicating matching residues. The default is to use the vertical | character for matching residues;

    DefaultAlignmentChars

    You can change the characters used in these displays using the MacVector | Preferences | Aligned Sequence pane. Note that these fields just accept a single character to be used in the alignment display;

    DefaultAlignmentCharsPane

    The “+1 or greater” field indicates the character used for matching characters using the currently selected scoring matrix. For DNA, this usually indicates identical matches, but for protein sequences this can be certain similar residues (leucine versus isoleucine for example).

    The “between -1 & +1” field is used for “neutral” matches – e.g. an “N” against a “G” for DNA. By default this is set to a space character.

    Finally, the “-1 or less” character is used for mismatches.

    Sometimes, you may be comparing sequences where you are far more interested in the mismatches between them than the matches. In the example above, there are mismatches in one section that do not immediately leap out form the page. Lets see what happens if we change the characters from the default to this;

    ModifiedAlignmentCharsPane

    After we click Apply, the alignment text window updates to make it much clearer where the mismatches lie;

    ModifiedAlignmentChars



    This is an article in a long running series of tips to help you get the most out of MacVector. If you want to get notified every time a new tip gets published, follow us @MacVector on twitter (or check the feed for the hashtag #101MacVectorTips) or like us on Facebook.

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    How Assembler uses quality scores to create assemblies

    By Chris | Published: December 7, 2015

    A common problem with all types of sequence assembly is distinguishing between sequencing errors and true genomic variations. Quality scores are one way to help the algorithm identify if a variation is of high quality and therefore likely to be a SNP or a sequencing error.

    For Assembler trace files can be basecalled with Phred, which adds quality data. Reads that are added in FASTQ format should already contain Phred quality scores. Both Phrap and Bowtie2 are phred quality score aware. However, Velvet does not currently use the quality scores when generating an assembly.

    For assembly with Phrap when the consensus is calculated it uses the quality scores as guides to how strongly to consider that particular sequence.  A single high scoring sequence will always contribute far higher to a consensus calculation then multiple low scoring ones.  Especially ones that have a score below 20 (shown in red). The scoring algorithm is logarithmic.  A score of 10 will mean that Phred has determined there to be a 1 in 10 chance that the basecall is in error.  1 in 20 means a 1 in 100 chance of an error.  20 and above is generally considered as acceptable, and so Assembler gives all scores above 20 as green (this cutoff point is different for the overall contig quality scores, as 40 or above is considered an acceptable score here).

    Since it’s Phrap that determines the consensus and the quality of the consensus, and it basically ignores poor quality sequence if there is good quality sequence at the same position, then you should not need to remove poor quality sequence. In fact, the Phrap manual specifically states that you should never remove poor quality sequence.  So a red scoring sequence will not contribute to the consensus.

    However, you can manually override the quality score in the contig editor.  The quality scores of 98 and 99 are reserved for manual editing of a trace file. So if you change any of a trace sequence using a valid IUPAC symbol, then the quality score will change to 99 and it will also change to be blue.  So if you are confident a particular sequence is correct, then overwriting it with the same symbol will change the quality score.  When calculating the sequence consensus, Assembler will always give special priority to manually edited traces with quality scores of 99. If there are 1 or more reads with manually edited bases, then the consensus will only be calculated from these, and the others will be ignored.  If there are multiple reads with manually edited, but non-matching, bases at the same point, then the consensus will use an ambiguity character to show this.

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