General musings from the MacVector team about sequence analysis, molecular biology, the Mac in general and of course your favorite sequence analysis app for the Mac!

In the first article in this series we took an overview of all the primer design tools in MacVector. In this second look at primer design in MacVector I’m going to look in more detail at our latest tool – Design Primers (Primer3).

Primer3 is a powerful, highly configurable command line tool for designing primers. However, using it from the command line is fairly complicated, and it has a hard learning curve. For example you need to supply your template sequence and parameters using a pre-XML data exchange format called BoulderIO. The output is also in this format. Consequently quite a few web interfaces have been developed to make Primer3 more accessible to users. MacVector also hides all this complication from you and supplies a very easy to use point and click interface to Primer3. It provides an exceedingly easy way to design a set of primers. Our aim in designing the interface was to allow a user to be able to design a set of primers to amplify a certain part of their sequence with a maximum of three mouse clicks. This was achieved and the simplest way to design primers with MacVector is:

1 – Select a particular feature or region of a sequence by highlighting it.

2 – From the menu choose

Analyze | Primers | Primer Design (Primer3)

3 – click OK.

…and that’s it! The default primer design settings will be used to produce the optimal primers to amplify that section.

The results are also presented in an easy to use way. You have a ranked list in spreadsheet form, that will allow you to quickly copy and paste your results into an email to send to your oligo synthesising service. You will also see a graphical map of the top five primer pairs along with the expected product that each primer pair would produce.

Our secondary design aim was to allow experienced users access to the powerful functionality of Primer3. For the default values we chose the settings that will give you the most sensible and useful primers in the majprity of scenarios. However, these can be changed in just a few mouse clicks. By modifying the advanced parameters it is possible to tune these values, to obtain the best primers for non-standard situations.

Design Primers also lets you have more control about where the primers are picked from. As well as selecting a single feature, you can also choose a Region to Scan . Here you can specify a region within which the amplified product should lie. Simply choose the size of product you want and Primer3 will design primers to amplify products of that size, from anywhere in that region. By default this method will design primers to produce products between 100 and 300 bases in length. However, you can change the minimum and maximum Product size values to design primers to produce shorter or longer products.

Finally, you can design primers by specifying Flanking Regions and your left and right primers will be selected from these. This is similar to the Amplify Feature/Region method but it is much more flexible, as you can specify more precisely the region that each primer is selected from.

In the next Primer Design article we’ll look at designing internal primers for realtime PCR.

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MacVector has had sequence assembly functionality for a long time. Many moons ago MacVector came with a OS9 only tool called AssemblyLIGN. Although a useful tool it was fairly limited and needed a complete replacement, so about five years ago Assembler was released, which is a much more modern tool for sequence assembly. In addition to Assembler MacVector also has built in assembly functionality. With the next release of MacVector (Which is version 11 and due out in the Summer) we will be introducing support for the assembly of short read data so I think it’s an appropriate time to review these existing sequence assembly tools.

Since MacVector’s main base of users are generally lab molecular biologists, so the built in tools are designed to be useful for their needs, and are focused on sequencing of known sequences. These needs are served by a function called Align to Reference, that allows you to take trace files and assemble them against a template sequence. Its an ideal tool if you are doing small scale sequencing projects. For example confirming a site directed mutagenesis experiment, checking that a construct you’ve just spent months doing has the insert in the correct order, or confirming the sequence of a cloned PCR fragment.

However, the functionality of this tool does not stop there and it’s also an excellent tool for SNP analysis, with some special tools to allow you to easily spot mutations from your original template sequence. Align To Ref is also designed for aligning mRNA/cDNA sequences against genomic templates to allow you to find intron/exon junctions. Other uses include taking EST sequences and again aligning them against a genomic template. That will show coding regions very nicely.

Then we come back to Assembler. This is intended for full scale de novo sequencing projects. Although available as a separate product, Assembler integrates completely into MacVector, and in fact allows you to run standard MacVector sequence analysis jobs directly against a contig as if it was a single sequence. That’s great for designing primers to amplify part of your contig for example. Assembler uses the Phred, Phrap and Cross Match algorithms to assembles traces into contigs. It shows full quality scores of the reads and the aligned contigs. Although it performs automatic vector trimming, you can also run a library of your usual cloning vectors against traces before assembling them to improve accuracy. Assembler will also basecall traces with a higher accuracy that many sequencing machines themselves can achieve. Incidentally the trial version of MacVector also includes the Assembler plugin. Just go to FILE – NEW – ASSEMBLY PROJECT to start Assembler.

I’ll write about the next release of Assembler and its improvements soon.