csPCR: A computational tool for the simulation of the Polymerase Chain Reaction

Dasu S, Williams A, Fofanov Y, Putonti C., csPCR: A computational tool for the simulation of the Polymerase Chain Reaction, Online J Bioinformatics, 11 (1): 30-33, 2010. Herein we present a computational simulation package PCR (csPCR) which models the PCR reaction, taking into consideration the issues of specificity, sensitivity, potential mishybridizations throughout the primer sequence as well as at the 3’ end, and primer interactions, including selfcomplementarity and primer-primer interactions. A single target sequence or multiple target sequences can be considered simultaneously in addition to multiple primer sequences; thus a complex community and/or a multiplex assay can be simulated in a manner analogous with the actual experiment. This tool leaves primer design to the user, as there is a wealth of existing programs already available, and rather focuses on simulation of the anticipated amplification and expected agarose gel in addition to providing information about the location(s) of amplification in both text and graphical format. The software is freely available at www.bioinfo.uh.edu/csPCR

Comparison of the Compositional Proclivities of the Complete Genomes of Plasmodium Falciparum and Human

Pathogens and hosts have a dynamic relationship, one that is ever changing at the molecular level - the pathogen influencing the evolutionary path of the host and the host influencing the evolutionary path of the pathogen. The pathogen’s adaptation to a particular host could serve several purposes, e.g. to mimic the host to avoid detection, to take advantage of the host’s cellular machinery, to increase virulence, etc. Recognizing these adaptations is far from trivial, particularly when the size of the pathogen’s and host’s genomes differ by orders of magnitudes. Novel algorithms and data structures have been developed in our laboratory that make it possible to quantify the “distance” (or number of mutations) separating pathogen and host sequences. Through the examination of these distances, we hypothesize that it is possible to monitor pathogen adaptations at the sequence level and further our understanding of the function of the pathogen machinery. Even though the genome of Plasmodium falciparum, the agent causing malaria in humans, is complete, the functions of many of the coding regions remain unknown. Herein we present the results of our exhaustive calculations for each of the annotated coding regions in P. falciparum and the human genom

Real-time qPCR Assay Development for Detection of Bacillus thuringiensis and Serratia marcescens DNA and the Influence of Complex Microbial Community DNA on Assay Sensitivity

Real-time quantitative polymerase chain reaction (real-time qPCR) assays are an effective technique to detect biological warfare agents and surrogate organisms. In my study, primers were designed to detect chromosomal DNA of biological warfare agent surrogates B. thuringiensis and S. marcescens (representing B. anthracis and Y. pestis, respectively) via real-time qPCR. Species-level specificity of the primers was demonstrated through comparisons with a bacterial strain panel and corroborated by qPCR data. Additionally, the primer efficacy was tested when template DNA was spiked into metagenomic DNA extracted from clinical lung microbiome samples. The results showed that while detection of B. thuringiensis or S. marcescens was still largely successful, the addition of metagenomic DNA did significantly inhibit amplification in most cases. The present study is significant not only for the design of multiple novel primer pairs able to detect bacterial agents in metagenomic DNA, but also the quantitative insight to the influence of background DNA on single species detection at low DNA concentrations

Microbial diversity of the Namib Desert salt pans

>Magister Scientiae - MScSalt pans are a characteristic feature of many dry deserts. The microbial communities inhabiting salt pans are thought to be particularly complex and are generally dominated by halophilic microorganisms. Although saline pools are frequently found within the hyper-arid Namib Desert, the microbial communities of these saline sites have been scarcely investigated. The aim of the present study was to characterise the archaeal, bacterial and cyanobacterial diversity inhabiting these extreme saline pools using three culture independent molecular techniques (DGGE, T-RFLP and 16S rRNA clone libraries). The physiochemical results, mainly the conductivity readings recorded from the sampling sites, indicated that the Gobabeb (103.0mS/cm) region was less saline than the two Swakopmund [(Sps01) (150.0mS/cm) and Sps02 (180.0mS/cm)] sites. Results obtained from DGGE and T-RFLP data were in agreement for both bacterial and cyanobacterial analysis indicating that the Gobabeb site was more diverse than the two Swakopmund sites (Sps01 and Sps02). In comparison, the archaeal community profiles for DGGE and T-RFLP analysis were in agreement illustrating that the archaeal community were more abundant in the two extreme Swakopmund saline sites. Phylogenetic data obtained from 16S rRNA gene clone libraries identified halophilic phylotypes (Rhodothermaceae, Idiomarinaceae Puniceicoccaceae and Cyanobacteria/Chloroplast, Family VII) normally associated with salt rich sites. In addition, a large number of unclassified taxa were identified. To conclude, the study highlighted the presence of a rich microbial diversity present within the salt pans of the Namib Desert and establishes a platform for future investigations.National Research Foundatio

Effect of the mutation rate and background size on the quality of pathogen identification

Motivation: Genomic-based methods have significant potential for fast and accurate identification of organisms or even genes of interest in complex environmental samples (air, water, soil, food, etc.), especially when isolation of the target organism cannot be performed by a variety of reasons. Despite this potential, the presence of the unknown, variable and usually large quantities of background DNA can cause interference resulting in false positive outcomes. Results: In order to estimate how the genomic diversity of the background (total length of all of the different genomes present in the background), target length and target mutation rate affect the probability of misidentifications, we introduce a mathematical definition for the quality of an individual signature in the presence of a background based on its length and number of mismatches needed to transform the signature into the closest subsequence present in the background. This definition, in conjunction with a probabilistic framework, allows one to predict the minimal signature length required to identify the target in the presence of different sizes of backgrounds and the effect of the target's mutation rate on the quality of its identification. The model assumptions and predictions were validated using both Monte Carlo simulations and real genomic data examples. The proposed model can be used to determine appropriate signature lengths for various combinations of target and background genome sizes. It also predicted that any genomic signatures will be unable to identify target if its mutation rate is > 5%. © The Author 2007. Published by Oxford University Press. All rights reserved