An efficient algorithm for minimal primer set selection

SUMMARY: We have developed U-PRIMER, a primer design program, to compute a minimal primer set (MPS) for any given set of DNA sequences. The U-PRIMER algorithm, which uses automatic variable fixing and automatic redundant constraint elimination to tackle the binary integer programming problem associated with the MPS selection problem. The program has been tested successfully with 32 adipocyte development-related genes and 9 TB-specific genes to obtain their respective MPSs. AVAILABILITY: A free copy of U-PRIMER implemented in C++ programming language is available from http://www.u-vision-biotech.com     

Multi-layered greedy network-growing algorithm: extension of greedy network-growing algorithm to multi-layered networks

In this paper, we extend our greedy network-growing algorithm to multi-layered networks. With multi-layered networks, we can solve many complex problems that single-layered networks fail to solve. In addition, the network-growing algorithm is used in conjunction with teacher-directed learning that produces appropriate outputs without computing errors between targets and outputs. Thus, the present algorithm is a very efficient network-growing algorithm. The new algorithm was applied to three problems: the famous vertical-horizontal lines detection problem, a medical data problem and a road classification problem. In all these cases, experimental results confirmed that the method could solve problems that single-layered networks failed to. In addition, information maximization makes it possible to extract salient features in input patterns.     

A greedy algorithm for aligning DNA sequences.

For aligning DNA sequences that differ only by sequencing errors, or by equivalent errors from other sources, a greedy algorithm can be much faster than traditional dynamic programming approaches and yet produce an alignment that is guaranteed to be theoretically optimal. We introduce a new greedy alignment algorithm with particularly good performance and show that it computes the same alignment as does a certain dynamic programming algorithm, while executing over 10 times faster on appropriate data. An implementation of this algorithm is currently used in a program that assembles the UniGene database at the National Center for Biotechnology Information.     

BranchClust: a phylogenetic algorithm for selecting gene families

Background  

Automated methods for assembling families of orthologous genes include those based on sequence similarity scores and those based on phylogenetic approaches. The first are easy to automate but usually they do not distinguish between paralogs and orthologs or have restriction on the number of taxa. Phylogenetic methods often are based on reconciliation of a gene tree with a known rooted species tree; a limitation of this approach, especially in case of prokaryotes, is that the species tree is often unknown, and that from the analyses of single gene families the branching order between related organisms frequently is unresolved.     

Phylogenetic diversity and the greedy algorithm

Given a phylogenetic tree with leaves labeled by a collection of species, and with weighted edges, the "phylogenetic diversity" of any subset of the species is the sum of the edge weights of the minimal subtree connecting the species. This measure is relevant in biodiversity conservation where one may wish to compare different subsets of species according to how much evolutionary variation they encompass. In this note we show that phylogenetic diversity has an attractive mathematical property that ensures that we can solve the following problem easily by the greedy algorithm: find a subset of the species of any given size k of maximal phylogenetic diversity. We also describe an extension of this result that also allows weights to be assigned to species.     

Turbo Tree: a fast algorithm for minimal trees

A branch and bound algorithm is described for searching rapidlyfor minimal length trees from biological data. The algorithmadds characters one at a time, rather than adding taxa, as inprevious branch and bound methods. The algorithm has been programmedand is available from the authors. A worked example is givenwith 33 characters and 15 taxa. About 8 x 10¹² binary treesare possible with 15 taxa but the branch and bound program findsthe minimal tree in <5 min on an IBM PC. Received on January 15, 1987; accepted on February 23, 1987     

Conserved PCR primer set designing for closely-related species to complete mitochondrial genome sequencing using a sliding window-based PSO algorithm

Background

Complete mitochondrial (mt) genome sequencing is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. For long template sequencing, i.e., like the entire mtDNA, it is essential to design primers for Polymerase Chain Reaction (PCR) amplicons which are partly overlapping each other. The presented chromosome walking strategy provides the overlapping design to solve the problem for unreliable sequencing data at the 5′ end and provides the effective sequencing. However, current algorithms and tools are mostly focused on the primer design for a local region in the genomic sequence. Accordingly, it is still challenging to provide the primer sets for the entire mtDNA.

Methodology/Principal Findings

The purpose of this study is to develop an integrated primer design algorithm for entire mt genome in general, and for the common primer sets for closely-related species in particular. We introduce ClustalW to generate the multiple sequence alignment needed to find the conserved sequences in closely-related species. These conserved sequences are suitable for designing the common primers for the entire mtDNA. Using a heuristic algorithm particle swarm optimization (PSO), all the designed primers were computationally validated to fit the common primer design constraints, such as the melting temperature, primer length and GC content, PCR product length, secondary structure, specificity, and terminal limitation. The overlap requirement for PCR amplicons in the entire mtDNA is satisfied by defining the overlapping region with the sliding window technology. Finally, primer sets were designed within the overlapping region. The primer sets for the entire mtDNA sequences were successfully demonstrated in the example of two closely-related fish species. The pseudo code for the primer design algorithm is provided.

Conclusions/Significance

In conclusion, it can be said that our proposed sliding window-based PSO algorithm provides the necessary primer sets for the entire mt genome amplification and sequencing.     

Finding minimal generating set for metabolic network with reversible pathways

Elementary flux modes give a mathematical representation of metabolic pathways in metabolic networks satisfying the constraint of non-decomposability. The large cost of their computation shifts attention to computing a minimal generating set which is a conically independent subset of elementary flux modes. When a metabolic network has reversible reactions and also admits a reversible pathway, the minimal generating set is not unique. A theoretical development and computational framework is provided which outline how to compute the minimal generating set in this case. The method is based on combining existing software to compute the minimal generating set for a “pointed cone” together with standard software to compute the Reduced Row Echelon Form.     

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

Aims: The gram‐positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results: A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one‐step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. Conclusions: The one‐step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. Significance and Impact of the Study: This work provides an efficient method for identification of lactococcal strains of industrial importance.     

Multiple sequence alignment using an exhaustive and greedy algorithm

We describe an exhaustive and greedy algorithm for improving the accuracy of multiple sequence alignment. A simple progressive alignment approach is employed to provide initial alignments. The initial alignment is then iteratively optimized against an objective function. For any working alignment, the optimization involves three operations: insertions, deletions and shuffles of gaps. The optimization is exhaustive since the algorithm applies the above operations to all eligible positions of an alignment. It is also greedy since only the operation that gives the best improving objective score will be accepted. The algorithms have been implemented in the EGMA (Exhaustive and Greedy Multiple Alignment) package using Java programming language, and have been evaluated using the BAliBASE benchmark alignment database. Although EGMA is not guaranteed to produce globally optimized alignment, the tests indicate that EGMA is able to build alignments with high quality consistently, compared with other commonly used iterative and non-iterative alignment programs. It is also useful for refining multiple alignments obtained by other methods.     

The genome of Mycobacterium leprae: a minimal mycobacterial gene set

Comparison of the recently sequenced genome of the leprosy-causing pathogen Mycobacterium leprae with other mycobacterial genomes reveals a drastic gene reduction and decay in M. leprae affecting many metabolic areas, exemplified by the retention of a minimal set of genes required for cell-wall biosynthesis.     

A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.     

A novel primer set for multilocus phylogenetic inference in East African cichlid fishes

The cichlid fishes in the East African Great Lakes are a prime model system for the study of adaptive radiation. Therefore, the availability of an elaborate phylogenetic framework is an important prerequisite. Previous phylogenetic hypotheses on East African cichlids are mainly based on mitochondrial and/or fragment‐based markers, and, to date, no taxon‐rich phylogeny exists that is based on multilocus DNA sequence data. Here, we present the design of an extensive new primer set (24 nuclear makers) for East African cichlids that will be used for multilocus phylogenetic analyses in the future. The primers are designed to work for both Sanger sequencing and next‐generation sequencing with the 454 technology. As a proof of principle, we validate these primers in a phylogenetically representative set of 16 cichlid species from Lake Tanganyika and main river systems in the area and provide a basic evaluation of the markers with respect to marker length and diversity indices.     

Evaluating rejuvenation of the forehead and brow: an algorithm for selecting the appropriate technique

The traditional reason for performing aesthetic surgery in the forehead and brow area has been to correct brow ptosis. However, there are several other conditions that may be improved by surgery in this area, including frown muscle imbalance, transverse forehead rhytids, and lateral brow laxity. Recently, a better understanding of the relevant anatomy and the evolving therapeutic modalities (including both open and closed techniques) have contributed to a renewed interest in aesthetic surgery in the forehead and brow area. One hundred consecutive patients were studied, each of whom underwent forehead rejuvenation for one of four indications-forehead rhytids, glabellar creases, lateral brow laxity, or brow ptosis. Thirty-eight percent of patients underwent open procedures, 30 percent underwent closed procedures, and 32 percent underwent limited procedures. Complications occurred in 4 percent of patients, including three patients who were dissatisfied with the surgery and one patient who required scar revision. Based on our findings, we formulated an algorithm that integrates the different indications and any concomitant procedures being performed. Our proposed treatment plan is based on this information. The algorithm may be used as a template when assessing a patient and adapting the recommended intervention to the individual patient.     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Biologically-generated primer for PCR: PCR primer of unknown sequence.

We describe a method for producing specific PCR primers directly from PCR product, bypassing the usual need to know the primer sequence. Lack of abundance of primers derived from a PCR product is compensated for by the incorporation of an arbitrary 5'TAG sequence which acts as a surrogate template target for the bulk amplification phase. We use the technique to amplify clonospecific rearranged immunoglobulin genes, which have applications as markers of lymphoid neoplasms for tracing the success of therapy. The principle may have wider application wherever conserved and variable regions of DNA are juxtaposed.