MacVector’s Primer Database allows you to save and retrieve primers from a Primer Database with the Primer3 and Quicktest Primer tools. 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 save a primer from Primer Design:
To use the Primer Database Search:
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 (e.g. InterProScan available in MacVector 15). 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.
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.
We wish you a happy, healthy and rewarding New Year!
The Analyze | Reverse Translation menu option lets you create a DNA sequence from a Protein sequence, reverse translated using a specific Genetic Code (by default, the Universal Genetic Code). The default option creates a DNA sequence with N’s and other ambiguities reflecting the degeneracy of the genetic code. This is great if you want to identify less ambiguous sections to design probes or primers and in fact MacVector will even display a list of probes with the least ambiguities.
However, MacVector also offers an optimization function if you are interested in designing a gene with codon usage optimized for expression in a particular organism.
To use this function, you do need to supply a codon usage table – a number of common tables are shipped with MacVector in the /Applications/MacVector/Codon Bias Tables/ directory. There are four different algorithms that MacVector provides for optimizing codon usage;
All analysis results for an individual sequence are collected into a single tabbed result window to reduce window clutter. However, there are times when it is very convenient to have results displayed in side-by-side windows. For example, if you run a dot plot you can zoom in to view sections of the comparison by drag-selecting over a region of interest in the Matrix Plot tab and the Aligned Sequence tab will update to only display the text alignments across the new selection. Constantly toggling between the tabs to drill down to the region you are interested in (e.g. a potential splice site on a genome versus cDNA alignment) can be very frustrating.
All you need to do is to click on the title of a tab, hold down the mouse button, then drag the selected tab away from the parent window. When you let go of the mouse button, a new window will be created containing just that single tab.
Not only that, you can organize the tabs into multiple windows if you like. If you drag a tab from one window and drop it onto the tab bar of another window (this only works on the tab bar, you can’t drop on the content region of a window), then the tab will be added to the target window.
Give it a try and get your result windows under control!
UPDATE – November 8, 2016 – Although the official switch off date is not until Wednesday the 9th, Entrez and BLAST are NOT currently working from MacVector 15.0.3 and earlier. We suspect this change is now permanent.
UPDATE – November 7, 2016 – We have just been notified by the NCBI that they will be making the server changes that will prevent Entrez and BLAST from working on any version from MacVector 15.0 and earlier this Wednesday, 9th November, NOT the 1st of December as we were previously told.
The NCBI hosts one of the world’s definitive sequence repositories and MacVector gives you direct access to sequences from these databases. Unfortunately, due to recently announced major infrastructure changes at the NCBI, MacVector’s ability to access these services will be severely impacted from December of this year. These changes are completely out of our control, however, our developers have been hard at work to resolve this. We are pleased to announce that we are about to release a new version that will restore this functionality: MacVector 15.1.
MacVector gives you access to the NCBI’s Entrez database. This allows you to search for and retrieve sequences directly to your desktop. Both simple and complex queries are allowed. For example, you can search for all human kinases and specific accession numbers.
MacVector also allows you to perform Blast searches directly from your desktop against the NCBI’s BLAST database. From example, you can submit a protein or gene sequences against published sequence databases, or even a reverse translated protein against DNA databases. You can very easily retrieve any hits directly to your Desktop as well as view the alignments.
Both of these tools directly access the NCBI’s databases via services that the NCBI offer.
From September to December the NCBI are making two major infrastructure changes that will impact MacVector’s use of these two services.
In September the NCBI are moving all their web servers to HTTPS only and in December they are switching off the HTTP web servers.
These changes have been implemented in a US government wide change to ensure safe access of websites for all. HTTP is insecure and HTTPS is much safer. The US government website explains this further.
From September onwards, they are gradually transitioning the way sequences are referenced, and therefore retrieved. Previously upon submission every sequence was assigned a GI number (since 1994) as well as an accession number. An accession number never changes, but with a new version of a sequence submission the GI number would change. From September 2016, all new sequence submissions will be assigned a single number instead. This will be the accession number AND version combined. The redundant GI number will no longer be assigned (although it will be a long time until older sequences have this replaced with the new version). The accession number and version will always be read together so you now have a simpler way of referencing a sequence, and more importantly, a human readable way of determining the version.
There are two toolkits that the NCBI offer for accessing these services from within a software application. NCBI Toolkit and Entrez Programming Utilities (E-Utils). The NCBI have released a new version of E-Utils that supports HTTPS connections and ACCESSION.VERSION numbering. However, NCBI Toolkit is not going to be updated. MacVector 15.0.3 uses NCBI Toolkit and this unfortunately means that from December MacVector 15.0.3 will no longer be able to access ENTREZ or Blast.
This change was announced with only a short notice and since the announcement our developers have been working extremely hard to migrate MacVector to use E-Utilities.
We’re pleased to announce that this will be released shortly. MacVector 15.1 contains a whole new implementation of the BLAST and Entrez tools. MacVector 15.1 will be released very shortly a few months before the final switching off of these services.
To avoid any interruption in service, please ensure that you have downloaded and installed MacVector 15.1 before the start of December.
Although our hand has been forced somewhat with the release of MacVector 15.1, we are still planning to release MacVector 15.5. Since the Blast and Entrez tools have now been rewritten, expect to see even more new enhancements to these tools in future releases.
You can very quickly annotate a region of interest in your sequence in the Editor tab. For example, showing introns in lower case or highlighting CDS features with a colored background.
To enter sequences as mixed case.
To color a region.
To change the case of a region.
[Edit December 20, 2017 – As of MacVector 15.5 you can simply right click a READ and select “SEE MATCHING READS” to view the pair of reads. The total sequence length is selected. ]
Insert length is the length of the sequence in between a pair of reads. Sequencers are supplied DNA samples in fragments of a known length and each end is sequenced (generally in a 5′ to 3′ direction from both ends).
For example if you have a fragment of 2Kbp and your reads had an average of 500bp, then the insert length would be around 1Kbp. Insert length is determined by the protocol that is used when preparing samples for sequencing. You should know the insert length (and orientation) of your sequencing data.
However, it may be that you do not know it. … or perhaps you have only a wide range and you want to see if using more stringent values would improve your assembly. Velvet will estimate this for you, however, Bowtie does not.
Here’s a quick way to estimate the insert length using MacVector and Assembler.
Now we will repeat the alignment with these extracted hits.
*If you are comfortable with working from the command line, then a quick step is to use “head” to extract 100 pairs.
A read in a FASTQ file is generally composed of four lines. The header, the sequence, a blank header and the quality line. So extracting a multiple of four lines would give you that number of reads.
If your pairs are in two files then run the following to extract 100 reads.
head -n 400 Mate1.fastq > Mate1_100reads.fastq head -n 400 Mate2.fastq > Mate2_100reads.fastq
Although there are two different ways to perform restriction enzyme analysis with MacVector, there are also additional places where restriction enzyme sites are shown. All these tools use the same set of restriction enzyme files to recognise enzymes. These files are updated regularly from the REBASE database.
The restriction enzymes are divided into multiple files. There’s the “Common Enzymes” file with commonly used enzymes. That is the default list for all tools. There are also files that are grouped by supplier or all known enzymes. However, if one of these does not suit, it is very easy to create your own set of enzymes. For example all enzymes that are stored in your lab’s freezer drawer!
For each tool you can use the same set of enzymes, or different files for each. So there are multiple ways to select which set of enzymes to use. For all tools you can also choose whether to SHOW ALL ENZYMES or if you have ONLY USE SELECTED ENZYMES checked, then click the OPEN button, ensure that your required sites are selected, and save the file.
..and do not forget that all restriction enzyme tools will also optionally show “one out” sites. These are restriction enzymes sites that can be introduced with a single substitution. QuickTest Primer will also show whether these are silent mutations as well. That is whether introducing that site will affect translation.
You can use the Analyze | Open Reading Frames function to very quickly find ORFs on a sequence. Did you know that you can very quickly turn those results into permanent CDS features on your sequence? After running the Open Reading Frames analysis, simply drag and drop the ORF objects you are interested in from the Results window onto the original sequence window. Here we are dropping the results into the Map tab but it works with any of the tabs.
Note that MacVector automatically fills out the /translation= qualifier for you with the predicted amino acid sequence. For optimum Genbank compliance, you should also manually add a /gene= qualifier with the preferred short name of the gene encoding your open reading frame and /product= with the full name of the product..