Discovery of primer pairs

In this example application we provide a command-line interface to the PD5 classes/methods to discover suitable primer pairs for amplification of a target region of a sequence, controlled by a variety of parameters. This command-line application is named pd5_cli and is available as part of the PD5 package. This serves mainly as a confirmation of the effectiveness of the PD5 library, and also provides an example for others who want to understand how to employ the PD5 library in their primer design applications. Furthermore, pd5_cli can be incorporated into a larger process or script to easily generate a large number of primers automatically without constant user intervention. The output is produced as plain text, HTML or CSV as required by the user. We have used this interface to generate 5127 sets of confirmation primers for seamless deletions of the ORFs in the S. pombe genome. These are available at http://www.aber.ac.uk/en/cs/research/cb/projects/pcr-primer-design/.

We have also provided a web interface PD5Web (http://PD5web.dcs.aber.ac.uk/), which allows the user to input their template sequence and chosen options. The website then uses pd5_cli to produce the primers and provides a web-based display of the results.

This command line application can also be used from within the Galaxy bioinformatics platform [14]–[16]. We have provided the necessary configuration file, and the PD5 wiki page describes the simple setup procedure (essentially, put PD5 folder as a subfolder of Galaxy's tools, and add a few lines to your Galaxy tools configuration file).

Design of large chimaeric primers for coupled PCR

PD5 is presently being used in the design of primer sets for the precise, seamless deletion of ORFs in the S. cerevisiae genome. For this work an application written in C/C⁺⁺ programming language has been developed making full use of the PD5 software library.

This method is based on three PCR reactions employing two different primer pairs to amplify regions of the yeast genome and to construct a URA3-based cassette. The position of one reverse primer is constrained to a fixed location on the template and so, to obtain a reasonable selection of candidates, the length of this primer can range from 30 to 60 bases, with an optimum of 40 bases. The forward primer is selected from a region 500–1000 bases upstream with similar length and GC content as the reverse primer.

Suitable primers for amplifying the URA3 marker gene are also required and these are concatenated with other sequences from the template to make long chimaeric primers. These long primers cannot be designed and optimised using any currently available primer analysis tools. However, the PD5 software library provides modules that are ideally suited to perform this task.

Furthermore, this application is required to automatically select optimum primers for several thousand PCRs without user intervention, and also to be processed using a distributed computer system. Again, the PD5 software library contains the necessary components, which are ideally suited for this.

It was this project that first inspired the development of PD5. Originally, we used Primer3 to find a selection of suitable primers, and BLAST, together with some supporting software, to detect secondary binding and secondary products. Primer3 could not analyse the long primers. We had problems with BLAST in that, to obtain the necessary sensitivity, we used shorter word sizes (4 and 5) and higher E values than recommended by NCBI. This dramatically increased processing time and produced many irrelevant hits, which required filtering by a BLAST file output parser, but still did not detect some significant secondary binding sites (the reasons for this are discussed in the Methods section). These problems were not exclusive to Primer3 and BLAST, hence the development of new primer design methods, which are now available in the PD5 software library.

The PD5 library solves several issues that are not addressed by other general purpose primer design software:

• The detection of potential secondary binding and secondary products was improved by using a sequence matching algorithm optimised for DNA sequences. These methods are included in the PD5 dna_find class.
• Existing methods for determining annealing temperature tend to be inaccurate when applied to long primers. To compensate for this, PD5 can simply generate primer pairs of similar length and GC content.
• The chimaeric primers used in the second PCR process can be up to 100 bases in length. The analysis of long primers such as these cannot be done with existing software and so the improved dimerisation analysis methods for long primers, included in PD5, proved invaluable.

The resulting code was executed on a Beowulf computer cluster using Condor [17] and produced primers that were used successfully to delete S. cerevisiae genes. In Information S1 (Section S1, Figures S1 and S2, Tables S1, S2 and S3) we provide a sample of the many PCR gel images that we have as evidence for the effectiveness of the primers that were designed for this work. The first gel image demonstrates good results from the first PCR stage for 32 distinct pairs of chimaeric primers targeting 32 different ORFs. This image is part of a larger collection detailing results for the seamless deletion technique, and the full set is to be published separately. The second gel image demonstrates good results for 12 pairs of primers designed to confirm the presence of 12 ORFs in a wild type strain of S. cerevisiae.

Microsatellite markers and flanking primers - PD5_ssr

The PD5 software modules can be integrated easily with other code where required to perform a primer design task. In this instance the task was to find microsatellite regions in the Miscanthus giganteus unassembled genome sequence data, and then to design flanking primers for each of these regions so that the microsatellites could be used as potential markers.

There are many existing software tools for the task of locating microsatellites (short tandem repeats of 2 to 6 nucleotides), for example, Sputnik (available from http://espressosoftware.com/sputnik/) and Misa [18]. Indeed, there are even software tools that already integrate the process of finding microsatellites and the process of finding their suitable flanking primers, for example BatchPrimer [7] and PrimerPro (available from http://webdocs.cs.ualberta.ca/yifeng/primerpro/). Our use of PD5 for this task serves the purpose of providing another illustrative use of this library. We demonstrate the flexibility of PD5 and show how it can be integrated with existing applications to facilitate the design of primers for analysing individual microsatellites in the Miscanthus genome. For this purpose we developed a C/C⁺⁺ application named PD5_ssr, which integrates PD5 with Sputnik. Sputnik is a public domain application for the discovery of microsatellites and it was chosen for its popularity, speed and simplicity.

We first made minor modifications to Sputnik to wrap it as a C⁺⁺ class, so that its previously hardcoded parameters can be easily changed, and so that multiple Sputnik instances can be created with different parameters. Then we begin by creating an instance of Sputnik with strict parameters, to search for high-scoring microsatellites. When a microsatellite region is found, the Sputnik code makes a call to PD5_ssr to start the search for flanking primers. During the search for primers PD5_ssr makes further calls to new instances of Sputnik (with different, more relaxed parameters) in order to make sure that the primers themselves and the flanking regions up to the microsatellite contain no other microsatellites. In this way the two pieces of software are closely intertwined, which is efficient and avoids the reading and writing of intermediate files, but their interaction remains restricted to just two function calls, and so the software remains maintainable and loosely coupled.

The sequence data comprised 21 Gb of short unassembled sequence reads (averaging approx 500 bases) from a Roche 454 next generation sequencer, equivalent to about three times coverage of the Miscanthus giganteus genome.

The constraints for the primer design for this application were as follows:

• Primers to be 20–30 bases in length, for economy.
• Product sizes to be between approximately 80 and 300 bases.
• Products should not contain Ns (frequently found in the reads).
• Primers to have the same GC content, with a preferred annealing temperature of as calculated using the oligoTm code incorporated from Primer3.
• Primers must not contain the microsatellite repeating unit.
• Primers and flanking regions must not contain other microsatellites.
• Primers must not have any non-specific binding within the same read fragment.

In total, 16124 microsatellite regions comprising at least 21 base pairs of repeating units of two, three or four bases, having flanking primers consistent with the above constraints, were produced by PD5_ssr for Miscanthus. Their utility is currently being evaluated experimentally. The full code for PD5_ssr is available as part of the PD5 package.

The PD5 software library

The overall modular class structure of PD5 is shown in Figure 1 where the main classes are on the left, the data classes are in the centre and the analysis classes are on the right. The main classes, primer and primer_pair, deal with the primer locations, primer candidate generation and primer candidate optimisation for individual primers or for pairs of primers. We expect most users to start with one of these two classes. After setting the various member variables to suit their needs they will call a function to generate candidates, followed by functions to calculate further properties of these candidates and then functions to sort and select the most optimal candidates.

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Figure 1. PD5 class structure.

The main classes are on the left, the data containment classes in the centre and the analysis classes on the right and below. Within each class only the main public methods and attributes (indicated by + prefix) are shown.

https://doi.org/10.1371/journal.pone.0080156.g001

The attributes in the data classes are those required for selection and optimisation of candidate primers. The data classes also contain methods for procuring the data from the relevant analysis class methods. The analysis classes are concerned with evaluating the potential for dimerisation, the numbers of binding sites and products, and the annealing temperatures for all candidate primers.

PD5 is designed such that specific classes can be accessed for specific lower level use. For example, the dimerisation class can be used in isolation to check if a particular primer pair might form a pair dimer, or a single instance of primer_data class can be used to test annealing temperature, self dimerisation, and hairpin formation for a given single primer. However, the ideal approach for the use of PD5 is that, from a single instance of the primer_pair class, the user has all methods available for efficiently designing primers. An example of this is given in Figure 2 showing how simple primer design can be achieved with little more than a handful of function/method calls. An example of more sophisticated primer design is given in Figure 3 showing more detailed design of an individual primer.

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Figure 2. Example code for basic primer analysis using the primer_pair class.

https://doi.org/10.1371/journal.pone.0080156.g002

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Figure 3. Example code for PD5_ssr, demonstrating some of the selection criteria for the forward primer.

https://doi.org/10.1371/journal.pone.0080156.g003

Hairpin formation and dimerisation

Potential dimerisation and hairpin formation are detected using rule based binding affinity methods very similar to those used by Primer3. However, this rule based method is applied to a sequence of a user definable length at the 3′ end of each primer and it is this modification that permits the detection of dimerisation in a much broader range of primer lengths than existing applications. The dimerisation and hairpin methods check the final bases at the 3′ tail for complementarity, and give a higher weighting to G and C complements, scoring up to a maximum . Parameters such as , and the weightings of the different bases can be altered to suit the user's needs.

Future plans for the PD5 project include research and development of thermodynamic methods for hairpin and dimerisation detection, the results of which are to be included in a future software release.

Annealing temperatures

The popular annealing temperature algorithms using thermodynamic data [19], [20] available in Primer3′s oligoTm code are included in the PD5 software library, and these have been shown to work well empirically for primers up to 32 bases in length. The annealing temperature class also includes several methods for calculating annealing temperatures using equations devised by Freier [21] and Wallace [22], which can calculate close approximations for longer primers. For the present, the default temperature calculation method used in the primer class is Primer3′s oligoTm method.

We aim to increase the range of annealing temperature algorithms available to PD5 in the near future. As an open source project, one of our objectives is to promote the inclusion of good code from other open source projects as reusable modules that increase collective knowledge and understanding of the most important factors that constrain the primer design process. One area of concern is that ideally, primers should have a more or less constant GC content throughout their length, but in instances where this is not possible the presence of many GC residues at the 3′ end can lead to mis-priming. Methods to determine the GC distribution along the length of primers would be beneficial particularly for use with longer primers. We provide an abstract base class for annealing temperature, and 4 derived classes that demonstrate how different temperature calculation algorithms can be incorporated into PD5. The end user can choose their preferred temperature method, but can easily change this for another method later, since all annealing temperature subclasses must implement the same core functionality. New temperature calculation methods can be added as further classes without disrupting existing code.

Secondary binding and products

PD5 presently has four methods for secondary binding detection. Three methods use third party software: BLAST [23], FASTA [24] and SSEARCH [25]. However, due to their heuristic nature, these methods will occasionally fail to detect significant alignments that can result in mis-priming and the corresponding production of alternative PCR products. This is because these applications are sequence alignment algorithms and work on the probability that a given sequence has significant sequence similarity to a matching sequence on the template. Potential primer binding sites are likely to have extremely varied probabilities of homology and sequence alignment algorithms could overlook primer binding sequences with a high probability of occurrence on the template. This is particularly a problem with small sequence stretches that have evenly spaced mismatches [11]. Furthermore, sequence alignment algorithms are computationally expensive compared with string matching algorithms and are largely unnecessary for the detection of secondary binding. Therefore, we developed a string matching algorithm optimised for the detection of primer binding on DNA sequences, which is included in the dna_find class. When tested on the S. cerevisiae genome and a plasmid sequence containing the URA3 gene from Kluyveromyces lactis, sequence alignment algorithms failed to detect 2% of the primer binding locations on the plasmid sequence, which were correctly detected by the sequence matching algorithm used in the secondary binding detection method in the dna_find class.

Secondary primer binding becomes a serious problem if it causes the exponential amplification of an alternative product. PD5 provides a method for PCR product detection. It uses the dna_find class to find all binding sites and then checks downstream from the location of each forward binding site for a reverse binding site within a user defined region on the template. The length of the user defined region depends essentially on the PCR extension time (approx. 1000 bases per min). The default length in PD5′s search_for_pcr_products method in the dna_find class is 3500 bases. This method detects all potential products. One of those products will, of course, be the target region, so it is the occurrence of more than one product that gives an indication of potential secondary product formation. A pseudocode algorithm outlining the basic operation of the product detection method is included in the Information S1(Section S2, Algorithm S1) along with the code and results of a demonstration application to find products in S. cerevisiae (Information S1, Section S2, Tables S4 and Figures S3 and S4).

To encourage further secondary binding methods to be added to PD5 we have implemented an abstract base class nsb, from which specific implementation classes can be derived, and dna_find provides one such example of this.

Multi-objective optimisation for primers

Multi-objective optimisation problems require an optimal solution to a problem that has characteristics that lie in several independent dimensions. For primer design, we prefer primers that are optimised for some or all of the previously described characteristics, such as suitable sizes and locations, no secondary binding and suitable annealing conditions. As is often the case, a solution that is optimal in all dimensions may not exist and in such cases, a Pareto optimal solution is chosen that takes into account a user specified weighting or priority on the dimensions deemed to be most important.

A multi-objective optimisation algorithm is included as a selectable option in the sort_candidates method in the primer class. The objective function is given by:(1)where is the user specified weighting and is a non-linear component applied to the value of the characteristic . The non-linear component we use is the sigmoid function given by:(2)where is a gain term used to adjust non-linearity of the sigmoid (default value 1.0), and is an offset used to push all characteristic scores into the non-linear region of the sigmoid that provides the best discrimination (). A brief example of non-linear multi-objective optimisation in included in the Information S1 (Section S3).

Determination of optimum primers

The determination of optimum primers is based on a stable iterative list sorting method where items at the bottom of the list can be rejected. A threshold value can be set so that all items in the list with characteristic values outside of the threshold can be eliminated from the list before re-sorting. This is particularly useful with rule based dimerisation scoring methods, where a score above a defined threshold indicates potential dimerisation. Primers in the sorting list with scores above the threshold are sorted to the bottom of the list and then rejected leaving a subset that contains only items with dimerisation scores below the threshold for subsequent sorting.

We currently use a prioritised sorting method where the priority for sorting according to each characteristic can be varied. This allows the sorting criteria to be changed, and therefore the order of the results, in accordance with the user's requirements. If, for example, the best forward primer we find has more than one binding site on the template, then there is the possibility of secondary product formation if the reverse primer has more than one binding site also. In such a circumstance we can increase the priority of the secondary binding score for the reverse primer so that those potential primers having just one template binding site are placed at the top of the list. In this way primer pairs less likely to make secondary products will be placed at the top of the list. Users who wish to use BLAST, FASTA or SSEARCH for the detection of potential secondary binding sites on the template will find this sorting approach useful to determine primer pairs that potentially form secondary products.

The multi-objective optimisation procedure described above is included in the options for candidate sorting and is best used as the last step in the sorting procedure. This allows for a more precise selection of the remaining candidate primers at the top of the lists previously sorted by the other options.

An application named basic_app is included in the PD5 download package that demonstrates the sorting procedure showing a list of primer candidates found, a list of primer candidates after sorting and the final list of candidate primer pairs.

Comparison with Primer3

Primer3 has far more options than PD5, with over 150 parameters, and a full evaluation/comparison of each of these is beyond the scope of this paper. Primer3 has been extensively used for over 13 years now, and has had many additions during this period. It has a large user base, from occasional non-experts using the web interface, to expert users who need specialist options. In this respect it easily provides a wide range of functionality that PD5 does not provide, including for example, allowing the user to specify that primers must span exon-exon junctions. The two systems share many common features, including methods for calculating annealing temperature, assessing dimer potential, and allowing the user to specify useful constraints such as the required GC composition. They also both provide a web interface, though PD5′s web interface currently has far fewer options. One of the main advantages that PD5 provides but Primer3 does not is an integrated non-specific binding search against a genome sequence. This search (dna_find) does not rely on external services such as BLAST, but is implemented natively, and it is not a heuristic search, so is guaranteed to find all alternative potential binding sites meeting the parameters the user has chosen. The other main focus of PD5 has been on the ability to design and test the properties of long primers, which are increasingly required by the synthetic biology community. This has involved the implementation of dimerisation and annealing temperature methods that are more suited to applications requiring long primers.