MCMC-ODPR : primer design optimization using Markov Chain Monte Carlo sampling

Background Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. Results After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. Conclusions MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base

Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments

Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens

An Iterative Beam Search Algorithm for Degenerate Primer Selection

Single Nucleotide Polymorphism (SNP) Genotyping is an important molecular genetics process in the early stages of producing results that will be useful in the medical field. Due to inherent complexities in DNA manipulation and analysis, many different methods have been proposed for a standard assay. One of the proposed techniques for performing SNP Genotyping requires amplifying regions of DNA surrounding a large number of SNP loci. In order to automate a portion of this particular method, it is necessary to select a set of primers for the experiment. Selecting these primers can be formulated as the Multiple Degenerate Primer Design (MDPD) problem. In this thesis, we describe an iterative beam-search algorithm, Multiple, It-erative Primer Selector (MIPS), for MDPD. Theoretical and experimental analyses show that this algorithm performs well compared to the limits of degenerate primer design. Furthermore, MIPS outperforms an existing algorithm which was designed for a related degenerate primer selection problem. Further analysis shows that, due to the composition of the human genome, the results from MIPS may not be realized in practice. Consequently, we address the challenges involved in selecting a suitable set of degenerate primers and possible future improvements to the algorithm

Parallel Architectures for Planetary Exploration Requirements (PAPER)

The Parallel Architectures for Planetary Exploration Requirements (PAPER) project is essentially research oriented towards technology insertion issues for NASA's unmanned planetary probes. It was initiated to complement and augment the long-term efforts for space exploration with particular reference to NASA/LaRC's (NASA Langley Research Center) research needs for planetary exploration missions of the mid and late 1990s. The requirements for space missions as given in the somewhat dated Advanced Information Processing Systems (AIPS) requirements document are contrasted with the new requirements from JPL/Caltech involving sensor data capture and scene analysis. It is shown that more stringent requirements have arisen as a result of technological advancements. Two possible architectures, the AIPS Proof of Concept (POC) configuration and the MAX Fault-tolerant dataflow multiprocessor, were evaluated. The main observation was that the AIPS design is biased towards fault tolerance and may not be an ideal architecture for planetary and deep space probes due to high cost and complexity. The MAX concepts appears to be a promising candidate, except that more detailed information is required. The feasibility for adding neural computation capability to this architecture needs to be studied. Key impact issues for architectural design of computing systems meant for planetary missions were also identified

Static Pricing Problems under Mixed Multinomial Logit Demand

Price differentiation is a common strategy for many transport operators. In this paper, we study a static multiproduct price optimization problem with demand given by a continuous mixed multinomial logit model. To solve this new problem, we design an efficient iterative optimization algorithm that asymptotically converges to the optimal solution. To this end, a linear optimization (LO) problem is formulated, based on the trust-region approach, to find a "good" feasible solution and approximate the problem from below. Another LO problem is designed using piecewise linear relaxations to approximate the optimization problem from above. Then, we develop a new branching method to tighten the optimality gap. Numerical experiments show the effectiveness of our method on a published, non-trivial, parking choice model

Fitting the Phenomenological MSSM

We perform a global Bayesian fit of the phenomenological minimal supersymmetric standard model (pMSSM) to current indirect collider and dark matter data. The pMSSM contains the most relevant 25 weak-scale MSSM parameters, which are simultaneously fit using `nested sampling' Monte Carlo techniques in more than 15 years of CPU time. We calculate the Bayesian evidence for the pMSSM and constrain its parameters and observables in the context of two widely different, but reasonable, priors to determine which inferences are robust. We make inferences about sparticle masses, the sign of the $\mu$ parameter, the amount of fine tuning, dark matter properties and the prospects for direct dark matter detection without assuming a restrictive high-scale supersymmetry breaking model. We find the inferred lightest CP-even Higgs boson mass as an example of an approximately prior independent observable. This analysis constitutes the first statistically convergent pMSSM global fit to all current data.Comment: Added references, paragraph on fine-tunin

PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data.

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity

Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA

BACKGROUND: The amplification of variable regions of immunoglobulins has become a major challenge in the cloning of antibody genes, whether from hybridoma cell lines or splenic B cells. Using conventional protocols, the heavy-chain variable region genes often are not amplified successfully from the hybridoma cell lines. RESULTS: A novel method was developed to design the degenerated primer of immunoglobulin cDNA and to amplify cDNA ends rapidly. Polymerase chain reaction protocols were performed to recognize the VH gene from the hybridoma cell line. The most highly conserved region in the middle of the VH regions of the Ig cDNA was identified, and a degenerated 5'primer was designed, using our algorithms. The VH gene was amplified by both the 3'RACE and 5'RACE. The VH sequence of CSA cells was 399 bp. CONCLUSION: The new protocol rescued the amplifications of the VH gene that had failed under conventional protocols. In addition, there was a notable increase in amplification specificity. Moreover, the algorithm improved the primer design efficiency and was shown to be useful both for building VH and VL gene libraries and for the cloning of unknown genes in gene families

Optimizing PCR primers targeting the bacterial 16S ribosomal RNA gene

Abstract Background Targeted amplicon sequencing of the 16S ribosomal RNA gene is one of the key tools for studying microbial diversity. The accuracy of this approach strongly depends on the choice of primer pairs and, in particular, on the balance between efficiency, specificity and sensitivity in the amplification of the different bacterial 16S sequences contained in a sample. There is thus the need for computational methods to design optimal bacterial 16S primers able to take into account the knowledge provided by the new sequencing technologies. Results We propose here a computational method for optimizing the choice of primer sets, based on multi-objective optimization, which simultaneously: 1) maximizes efficiency and specificity of target amplification; 2) maximizes the number of different bacterial 16S sequences matched by at least one primer; 3) minimizes the differences in the number of primers matching each bacterial 16S sequence. Our algorithm can be applied to any desired amplicon length without affecting computational performance. The source code of the developed algorithm is released as the mopo16S software tool (Multi-Objective Primer Optimization for 16S experiments) under the GNU General Public License and is available at http://sysbiobig.dei.unipd.it/?q=Software#mopo16S. Conclusions Results show that our strategy is able to find better primer pairs than the ones available in the literature according to all three optimization criteria. We also experimentally validated three of the primer pairs identified by our method on multiple bacterial species, belonging to different genera and phyla. Results confirm the predicted efficiency and the ability to maximize the number of different bacterial 16S sequences matched by primers

MICA: desktop software for comprehensive searching of DNA databases

BACKGROUND: Molecular biologists work with DNA databases that often include entire genomes. A common requirement is to search a DNA database to find exact matches for a nondegenerate or partially degenerate query. The software programs available for such purposes are normally designed to run on remote servers, but an appealing alternative is to work with DNA databases stored on local computers. We describe a desktop software program termed MICA (K-Mer Indexing with Compact Arrays) that allows large DNA databases to be searched efficiently using very little memory. RESULTS: MICA rapidly indexes a DNA database. On a Macintosh G5 computer, the complete human genome could be indexed in about 5 minutes. The indexing algorithm recognizes all 15 characters of the DNA alphabet and fully captures the information in any DNA sequence, yet for a typical sequence of length L, the index occupies only about 2L bytes. The index can be searched to return a complete list of exact matches for a nondegenerate or partially degenerate query of any length. A typical search of a long DNA sequence involves reading only a small fraction of the index into memory. As a result, searches are fast even when the available RAM is limited. CONCLUSION: MICA is suitable as a search engine for desktop DNA analysis software