UniPrimer: A Web-Based Primer Design Tool for Comparative Analyses of Primate Genomes

Whole genome sequences of various primates have been released due to advanced DNA-sequencing technology. A combination of computational data mining and the polymerase chain reaction (PCR) assay to validate the data is an excellent method for conducting comparative genomics. Thus, designing primers for PCR is an essential procedure for a comparative analysis of primate genomes. Here, we developed and introduced UniPrimer for use in those studies. UniPrimer is a web-based tool that designs PCR- and DNA-sequencing primers. It compares the sequences from six different primates (human, chimpanzee, gorilla, orangutan, gibbon, and rhesus macaque) and designs primers on the conserved region across species. UniPrimer is linked to RepeatMasker, Primer3Plus, and OligoCalc softwares to produce primers with high accuracy and UCSC In-Silico PCR to confirm whether the designed primers work. To test the performance of UniPrimer, we designed primers on sample sequences using UniPrimer and manually designed primers for the same sequences. The comparison of the two processes showed that UniPrimer was more effective than manual work in terms of saving time and reducing errors

PRISE2: software for designing sequence-selective PCR primers and probes.

BackgroundPRISE2 is a new software tool for designing sequence-selective PCR primers and probes. To achieve high level of selectivity, PRISE2 allows the user to specify a collection of target sequences that the primers are supposed to amplify, as well as non-target sequences that should not be amplified. The program emphasizes primer selectivity on the 3' end, which is crucial for selective amplification of conserved sequences such as rRNA genes. In PRISE2, users can specify desired properties of primers, including length, GC content, and others. They can interactively manipulate the list of candidate primers, to choose primer pairs that are best suited for their needs. A similar process is used to add probes to selected primer pairs. More advanced features include, for example, the capability to define a custom mismatch penalty function. PRISE2 is equipped with a graphical, user-friendly interface, and it runs on Windows, Macintosh or Linux machines.ResultsPRISE2 has been tested on two very similar strains of the fungus Dactylella oviparasitica, and it was able to create highly selective primers and probes for each of them, demonstrating the ability to create useful sequence-selective assays.ConclusionsPRISE2 is a user-friendly, interactive software package that can be used to design high-quality selective primers for PCR experiments. In addition to choosing primers, users have an option to add a probe to any selected primer pair, enabling design of Taqman and other primer-probe based assays. PRISE2 can also be used to design probes for FISH and other hybridization-based assays

MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research

Background: Methylation-sensitive restriction enzymes—polymerase chain reaction (MSRE-PCR) has been used in epigenetic research to identify genome-wide and gene-specific DNA methylation. Currently, epigenome-wide discovery studies provide many candidate regions for which the MSREqPCR approach can be very effective to confirm the findings. MSREqPCR provides high multiplexing capabilities also when starting with limited amount of DNA-like cfDNA to validate many targets in a time- and cost-effective manner. Multiplex design is challenging and cumbersome to define specific primers in an effective manner, and no suitable software tools are freely available for high-throughput primer design in a time-effective manner and to automatically annotate the resulting primers with known SNPs, CpG, repeats, and RefSeq genes. Therefore a robust, powerful, high-throughput, optimized, and methylation-specific primer design tool with great accuracy will be very useful.Results: We have developed a novel pipeline, called MSRE-HTPrimer, to design MSRE-PCR and genomic PCR primers pairs in a very efficient manner and with high success rate. First, our pipeline designs all possible PCR primer pairs and oligos, followed by filtering for SNPs loci and repeat regions. Next, each primer pair is annotated with the number of cut sites in primers and amplicons, upstream and downstream genes, and CpG islands loci. Finally, MSRE-HTPrimer selects resulting primer pairs for all target sequences based on a custom quality matrix defined by the user. MSRE-HTPrimer produces a table for all resulting primer pairs as well as a custom track in GTF file format for each target sequence to visualize it in UCSC genome browser.Conclusions: MSRE-HTPrimer, based on Primer3, is a high-throughput pipeline and has no limitation on the number and size of target sequences for primer design and provides full flexibility to customize it for specific requirements. It is a standalone web-based pipeline, which is fully configured within a virtual machine and thus can be readily used without any configuration. We have experimentally validated primer pairs designed by our pipeline and shown a very high success rate of primer pairs: out of 190 primer pairs, 71 % could be successfully validated. The MSRE-HTPrimer software is freely available from http://sourceforge.net/p/msrehtprimer/wiki/Virtual_Machine/ as a virtual machine

Applications in computer-assisted biology

Biology is becoming a data-rich science driven by the development of high-throughput technologies like next-generation DNA sequencing. This is fundamentally changing biological research. The genome sequences of many species are becoming available, as well as the genetic variation within a species, and the activity of the genes in a genome under various conditions. With the opportunities that these new technologies offer, comes the challenge to effectively deal with the large volumes of data that they produce. Bioinformaticians have an important role to play in organising and analysing this data to extract biological information and gain knowledge. Also for experimental biologists computers have become essential tools. This has created a strong need for software applications aimed at biological research. The chapters in this thesis detail my contributions to this area. Together with molecular biologists, plant breeders, immunologists, and microbiologists, I have developed several software tools and performed computational analyses to study biological questions. Chapter 2 is about Primer3Plus, a web tool that helps biologists to design DNA primers for their experiments. These primers are typically short stretches of DNA (~20 nucleotides) that direct the DNA replication machinery to copy a selected region of a DNA molecule. The specificity of a primer is determined by several chemical and physical properties and therefore designing good primers is best done with the help of a computer program. Primer3Plus offers a user-friendly task-oriented web interface to the popular primer3 primer design program. Primer3Plus clearly fulfils a need in the biological research community as already over 400 scientific articles have cited the Primer3Plus publication. Single nucleotide differences or polymorphisms (SNPs) that are present within a species can be used as markers to link phenotypic observations to locations on the genome. Chapter 3 discusses QualitySNPng, which is a stand-alone software tool for finding SNPs in high-throughput sequencing data. QualitySNPng was inspired by the QualitySNP pipeline for SNP detection that was published in 2006 and it uses similar filtering criteria to distinguish SNPs from technical artefacts like sequence read errors. In addition, the SNPs are used to predict haplotypes. QualitySNPng has a graphical user interface that allows the user to run the SNP detection and evaluate the results. It has already been successfully used in several projects on marker detection for plant breeding. Single nucleotide polymorphisms can lead to single amino acid changes in protein sequences. These single amino acid polymorphisms (SAPs) play a key role in graft-versus-host (GVH) effects that often accompany tissue transplantations. A beneficial variant of GVH is the graft-versus-leukaemia (GVL) effect that is sometimes witnessed after bone marrow transplantation in leukaemia patients. When the GVL effect occurs, the donor’s immune cells actively destroy residual tumour cells in the patient. The GVL effect can already be elicited by a single amino acid difference between the patient and the donor. Currently, a small number of SAPs that can elicit a GVL effect are known and these are used to select the right bone marrow donor for a leukaemia patient. Together with researchers at the Leiden University Medical Center I developed a database to aid in the discovery of more such SAPs. We called this database the “Human Short Peptide Variation database” or HSPVdb. It is described in chapter 4. The work described in chapter 5 is focused on the regions in bacterial genomes that are involved in gene regulation, the promoters. Intrigued by anecdotal evidence that duplication of bacterial promoters can activate or silence genes, we investigated how often promoter duplication occurs in bacterial genomes. Using the large number of bacterial genomes that are currently available, we looked for clusters of highly similar promoter regions. Since duplication assumes some sort of mobility, we termed the duplicated promoters: putative mobile promoters or PMPs. We found over 4,000 clusters of PMPs in 1,043 genomes. Most of the clusters consist of two members, indicating a single duplication event, but we also found much larger clusters of PMPs within some genomes. A number of PMPs are present in multiple species, even in very distantly related bacterial species, suggesting perhaps that these were subjected to horizontal gene transfer. The mobile promoters could play an important role in the rapid rewiring of gene regulatory networks. Chapter 6 discusses how current biological research can adapt to make full use of the opportunities offered by the high-throughput technologies by following three different approaches. The first approach empowers the biologists with user-friendly software that allows him to analyse the large volumes of genome scale data without requiring expert computer skills. In the second approach the biologist teams up with a bioinformatician to combine in-depth biological knowledge with expert computational skills. The third approach combines the biologist and the bioinformatician in one person by teaching the biologist computational skills. Each of these three approaches has it merits and shortcomings, so I do not expect any of them to become dominant in the near future. Looking further ahead, it seems inevitable that any biologist will have to learn at least the basics of computational methods and that this should be an integral part of biology education. Bioinformatics might in time cease to exist as a separate field and instead become an intrinsic aspect of most biological research disciplines.</p

MultiPriDe: automated batch development of quantitative real-time PCR primers

Quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) is a commonly employed gene expression quantification technique. This requires the development of appropriately targeted oligonucleotide primers, which necessitates the identification of ideal amplicons, development of optimized oligonucleotide sequences under most favorable pre-determined reaction conditions, and management of the resultant target-oligonucleotide pair information for each gene to be studied. The Primer3 utility exists for development of oligonucleotide primers and fills that role effectively. However, the manual process of identifying target sites and individually generating primers is inefficient and prone to user-introduced error, especially when a large number of genes are to be examined. We have developed MultiPriDe (Multiple Primer Design), a Perl utility that accepts batch lists of Gene database identifiers, collects available intron and exon position data critical to qRT–PCR primer development, and supplies these sites as identified targets for the Primer3 utility. This automated ‘gene to primer’ procedure is coupled with a set of optimized hybridization conditions used by the Primer3 utility to maximize successful primer design. MultiPriDe and assembled repeat libraries are available upon request. Please direct requests to [email protected]

mGenomeSubtractor: a web-based tool for parallel in silico subtractive hybridization analysis of multiple bacterial genomes

mGenomeSubtractor performs an mpiBLAST-based comparison of reference bacterial genomes against multiple user-selected genomes for investigation of strain variable accessory regions. With parallel computing architecture, mGenomeSubtractor is able to run rapid BLAST searches of the segmented reference genome against multiple subject genomes at the DNA or amino acid level within a minute. In addition to comparison of protein coding sequences, the highly flexible sliding window-based genome fragmentation approach offered can be used to identify short unique sequences within or between genes. mGenomeSubtractor provides powerful schematic outputs for exploration of identified core and accessory regions, including searches against databases of mobile genetic elements, virulence factors or bacterial essential genes, examination of G+C content and binucleotide distribution bias, and integrated primer design tools. mGenomeSubtractor also allows for the ready definition of species-specific gene pools based on available genomes. Pan-genomic arrays can be easily developed using the efficient oligonucleotide design tool. This simple high-throughput in silico ‘subtractive hybridization’ analytical tool will support the rapidly escalating number of comparative bacterial genomics studies aimed at defining genomic biomarkers of evolutionary lineage, phenotype, pathotype, environmental adaptation and/or disease-association of diverse bacterial species. mGenomeSubtractor is freely available to all users without any login requirement at: http://bioinfo-mml.sjtu.edu.cn/mGS/

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/

QuantPrime – a flexible tool for reliable high-throughput primer design for quantitative PCR

<p>Abstract</p> <p>Background</p> <p>Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.</p> <p>Results</p> <p>Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet <url>http://www.quantprime.de/</url> or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant <it>Arabidopsis thaliana</it>, the crop <it>Hordeum vulgare </it>and the model green alga <it>Chlamydomonas reinhardtii </it>show success rates of designed primer pairs exceeding 96%.</p> <p>Conclusion</p> <p>QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative <it>in situ </it>hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays.</p