Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis

AbstractThe polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator

ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples

Haplotype affinities resolve a major component of goat (<i>Capra hircus</i>) MtDNA D-loop diversity and reveal specific features of the Sardinian stock

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using Bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity. We identified haplotype groups overrepresented in Sardinian goats as a result of founder effects. We found that breeders maintain diversity of matrilines most likely through equalization of the reproductive potential. Moreover, the relevant amount of inter-farm mtDNA diversity found does not increase proportionally with distance. Our results illustrate the effects of breeding practices on the composition of maternal gene pool and identify mtDNA types that may be considered in projects aimed at retrieving the maternal component of the oldest breeds of Sardinia.</br

Cryptic diversity within the major trypanosomiasis vector Glossina fuscipes revealed by molecular markers

Background: The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units. Principal Findings: The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f.fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled fromEthiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations. Conclusion/Significance: We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme

Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)

.Rotaviruses are a major cause of acute gastroenteritis, which is often fatal in infants. The viral genome consists of 11 double-stranded RNA segments, but little is known about their cis-acting sequences and structural elements. Covariation studies and phylogenetic analysis exploring the potential structure of RNA11 of rotaviruses suggested that, besides the previously predicted "modified panhandle" structure, the 5' and 3' termini of one of the isoforms of the bovine rotavirus UKtc strain may interact to form a tRNA-like structure (TRLS). Such TRLSs have been identified in RNAs of plant viruses, where they are important for enhancing replication and packaging. However, using tRNA mimicry assays (in vitro aminoacylation and 3'- adenylation), we found no biochemical evidence for tRNA-like functions of RNA11. Capping, synthetic 3' adenylation and manipulation of divalent cation concentrations did not change this finding. NMR studies on a 5'- and 3'-deletion construct of RNA11 containing the putative intra-strand complementary sequences supported a predominant panhandle structure and did not conform to a cloverleaf fold despite the strong evidence for a predicted structure in this conserved region of the viral RNA. Additional viral or cellular factors may be needed to stabilise it into a form with tRNA-like properties

Sequence Search Algorithms for Single Pass Sequence Identification: Does One Size Fit All?

Bioinformatic tools have become essential to biologists in their quest to understand the vast quantities of sequence data, and now whole genomes, which are being produced at an ever increasing rate. Much of these sequence data are single-pass sequences, such as sample sequences from organisms closely related to other organisms of interest which have already been sequenced, or cDNAs or expressed sequence tags (ESTs). These single-pass sequences often contain errors, including frameshifts, which complicate the identification of homologues, especially at the protein level. Therefore, sequence searches with this type of data are often performed at the nucleotide level. The most commonly used sequence search algorithms for the identification of homologues are Washington University’s and the National Center for Biotechnology Information's (NCBI) versions of the BLAST suites of tools, which are to be found on websites all over the world. The work reported here examines the use of these tools for comparing sample sequence datasets to a known genome. It shows that care must be taken when choosing the parameters to use with the BLAST algorithms. NCBI’s version of gapped BLASTn gives much shorter, and sometimes different, top alignments to those found using Washington University’s version of BLASTn (which also allows for gaps), when both are used with their default parameters. Most of the differences in performance were found to be due to the choices of default parameters rather than underlying differences between the two algorithms. Washington University’s version, used with defaults, compares very favourably with the results obtained using the accurate but computationally intensive Smith–Waterman algorithm

Re-annotation of protein-coding genes in 10 complete genomes of Neisseriaceae family by combining similarity-based and composition-based methods.

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SNPServer: a real-time SNP discovery tool

SNPServer is a real-time flexible tool for the discovery of SNPs (single nucleotide polymorphisms) within DNA sequence data. The program uses BLAST, to identify related sequences, and CAP3, to cluster and align these sequences. The alignments are parsed to the SNP discovery software autoSNP, a program that detects SNPs and insertion/deletion polymorphisms (indels). Alternatively, lists of related sequences or pre-assembled sequences may be entered for SNP discovery. SNPServer and autoSNP use redundancy to differentiate between candidate SNPs and sequence errors. For each candidate SNP, two measures of confidence are calculated, the redundancy of the polymorphism at a SNP locus and the co-segregation of the candidate SNP with other SNPs in the alignment. SNPServer is available at