Ancient DNA analysis of the Japanese sea lion (Zalophus californianus japonicus Peters, 1866): preliminary results using mitochondrial control-region sequences

In this study, we successfully extracted ancient DNA from skeletal remains of the Japanese sea lion-a species that is practically extinct-from archaeological sites and determined a partial sequence of its mitochondrial DNA control region. A molecular phylogenetic tree constructed by the neighbor-joining (NJ) method showed that the sequences from Japanese sea lions clustered together, with a high bootstrap value, and that this cluster was closest to the California sea lion cluster. The distinctly divergent cluster of Japanese sea lions reflected the morphological classification of these animals as a distinct species of the genus Zalophus; however, proximity to the California sea lion cluster simultaneously implied conformation with the traditional classification of these animals as a subspecies of Zalophus californianus. The average amount of nucleotide substitution between the Japanese and California sea lions was 7.02%. The Japanese and California sea lions were estimated to have diverged 2.2 million years ago, i.e., in the late Pliocene Epoch. This is the first report on a genetic analysis of the Japanese sea lion.     

MitoBamAnnotator: A web-based tool for detecting and annotating heteroplasmy in human mitochondrial DNA sequences

The use of Next-Generation Sequencing of mitochondrial DNA is becoming widespread in biological and clinical research. This, in turn, creates a need for a convenient tool that detects and analyzes heteroplasmy. Here we present MitoBamAnnotator, a user friendly web-based tool that allows maximum flexibility and control in heteroplasmy research. MitoBamAnnotator provides the user with a comprehensively annotated overview of mitochondrial genetic variation, allowing for an in-depth analysis with no prior knowledge in programming.     

Web-based virtual microscopy for parasitology: a novel tool for education and quality assurance

Background

The basis for correctly assessing the burden of parasitic infections and the effects of interventions relies on a somewhat shaky foundation as long as we do not know how reliable the reported laboratory findings are. Thus virtual microscopy, successfully introduced as a histopathology tool, has been adapted for medical parasitology.

Methodology/Principal Findings

Specimens containing parasites in tissues, stools, and blood have been digitized and made accessible as a “webmicroscope for parasitology” (WMP) on the Internet (http://www.webmicroscope.net/parasitology).These digitized specimens can be viewed (“navigated” both in the x-axis and the y-axis) at the desired magnification by an unrestricted number of individuals simultaneously. For virtual microscopy of specimens containing stool parasites, it was necessary to develop the technique further in order to enable navigation in the z plane (i.e., “focusing”). Specimens were therefore scanned and photographed in two or more focal planes. The resulting digitized specimens consist of stacks of laterally “stiched” individual images covering the entire area of the sample photographed at high magnification. The digitized image information (∼10 GB uncompressed data per specimen) is accessible at data transfer speeds from 2 to 10 Mb/s via a network of five image servers located in different parts of Europe. Image streaming and rapid data transfer to an ordinary personal computer makes web-based virtual microscopy similar to conventional microscopy.

Conclusion/Significance

The potential of this novel technique in the field of medical parasitology to share identical parasitological specimens means that we can provide a “gold standard”, which can overcome several problems encountered in quality control of diagnostic parasitology. Thus, the WMP may have an impact on the reliability of data, which constitute the basis for our understanding of the vast problem of neglected tropical diseases. The WMP can be used also in the absence of a fast Internet communication. An ordinary PC, or even a laptop, may function as a local image server, e.g., in health centers in tropical endemic areas.     

Computer programs for the analysis and the management of DNA sequences.

A program package is described for the management and the analysis of DNA sequence data. The programs - with the exception of a few Fortran routines - are written in the programming language APL. They are best used interactively although batch processing is possible. The package has been in constant use for about 3 years and contains programs for most of the routine problems presently found in a DNA sequencing laboratory.     

Fourier and wavelet transform analysis, a tool for visualizing regular patterns in DNA sequences

A correlation function that compares each base in a DNA sequence to its various neighbours and which is subsequently processed by Fourier and wavelet transforms has been developed. The procedure has been applied to sequences from the human chromosome 22, to nef genes from various HIV clones and to myosin heavy chain DNA. It permits to readily visualize regular features in DNA which are related to the stability of heteroduplexes formed upon strand slippage.     

AlignMiner: a Web-based tool for detection of divergent regions in multiple sequence alignments of conserved sequences

Background  

Multiple sequence alignments are used to study gene or protein function, phylogenetic relations, genome evolution hypotheses and even gene polymorphisms. Virtually without exception, all available tools focus on conserved segments or residues. Small divergent regions, however, are biologically important for specific quantitative polymerase chain reaction, genotyping, molecular markers and preparation of specific antibodies, and yet have received little attention. As a consequence, they must be selected empirically by the researcher. AlignMiner has been developed to fill this gap in bioinformatic analyses.     

ChromaPipe: a pipeline for analysis, quality control and management for a DNA sequencing facility

Optimizing and monitoring the data flow in high-throughput sequencing facilities is important for data input and output, for tracking the status of results for the users of the facility, and to guarantee a good, high-quality service. In a multi-user system environment with different throughputs, each user wants to access his/her data easily, track his/her sequencing history, analyze sequences and their quality, and apply some basic post-sequencing analysis, without the necessity of installing further software. Recently, Fiocruz established such a core facility as a "technological platform". Infrastructure includes a 48-capillary 3730 DNA Sequence Analyzer (Applied Biosystems) and supporting equipment. The service includes running samples for large-scale users, performing DNA sequencing reactions and runs for medium and small users, and participation in partial or full genome projects. We implemented a workflow that fulfills these requirements for small and high throughput users. Our implementation also includes the monitoring of data for continuous quality improvement (reports by plate, month and user) by the sequencing staff. For the user, different analyses of the chromatograms, such as visualization of good quality regions, as well as processing, such as comparisons or assemblies, are available. So far, 180 users have made use of the service, generating 155,000 sequences, 35% of which were produced for the BCG Moreau-RJ genome project. The pipeline (named ChromaPipe for Chromatogram Pipeline) is available for download by the scientific community at the url http://bioinfo.pdtis.fiocruz.br/ChromaPipe/. The support for assembly is also configured as a web service: http://bioinfo.pdtis.fiocruz.br/Assembly/.     

Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly

The phylogenetic position of cyclostomes, i.e., the relationships between hagfishes, lampreys, and jawed vertebrates is an unresolved problem. Anatomical data support the paraphyly of cyclostomes, whereas nuclear genes data support monophyly of cyclostomes. Previous results obtained using mitochondrial DNA are ambiguous, presumably due to a lack of informative sequences. By adding the complete mtDNA of a hagfish, Eptatretus burgeri, we have generated a novel data set for sequences of hagfishes and of lampreys. The addition of this mtDNA sequence to the 12 taxa we have already used becomes sufficient to obtain unambiguous results. This data set, which includes sequences of mtDNA of animals closely related to the lamprey/hagfish node, was used in a phylogenetic analysis with two independent statistical approaches and unequivocally supported the monophyly of cyclostomes. Thus molecular data, i.e., our results and those obtained using nuclear genes, conclude that hagfishes and lampreys form a clade.     

REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences

REMA is an interactive web-based program which predicts endonuclease cut sites in DNA sequences. It analyses multiple sequences simultaneously and predicts the number and size of fragments as well as provides restriction maps. The users can select single or paired combinations of all commercially available enzymes. Additionally, REMA permits prediction of multiple sequence terminal fragment sizes and suggests suitable restriction enzymes for maximally discriminatory results. REMA is an easy to use, web based program which will have a wide application in molecular biology research. Availability: REMA is written in Perl and is freely available for non-commercial use. Detailed information on installation can be obtained from Jan Szubert (jan.szubert@gmail.com) and the web based application is accessible on the internet at the URL http://www.macaulay.ac.uk/rema Contact: b.singh@macaulay.ac.uk.     

SWORDS: A statistical tool for analysing large DNA sequences

In this article, we present some simple yet effective statistical techniques for analysing and comparing large DNA sequences. These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in public domain databases housed in the Internet, we demonstrate how SWORDS can be conveniently used by molecular biologists and geneticists to unmask biologically important features hidden in large sequences and assess their statistical significance.     

The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs.

Variation in the human mitochondrial genome (mtDNA) is now routinely described and used to infer the histories of peoples, by means of one of two procedures, namely, the assaying of RFLPs throughout the genome and the sequencing of parts of the control region (CR). Using 95 samples from the Near East and northwest Caucasus, we present an analysis based on both systems, demonstrate their concordance, and, using additional available information, present the most refined phylogeny to date of west Eurasian mtDNA. We describe and apply a nomenclature for mtDNA clusters. Hypervariable nucleotides are identified, and the relative mutation rates of the two systems are evaluated. We point out where ambiguities remain. The identification of signature mutations for each cluster leads us to apply a hierarchical scheme for determining the cluster composition of a sample of Berber speakers, previously analyzed only for CR variation. We show that the main indigenous North African cluster is a sister group to the most ancient cluster of European mtDNAs, from which it diverged approximately 50,000 years ago.     

Phylogenetic relationships of four endemic genera of the Phasianidae in China based on mitochondrial DNA control-region genes

The taxonomic status of some genera within the Phasianidae remains controversial. To demonstrate the phylogenetic relationships of four endemic genera (Tetraophasis, Ithaginis, Crossoptilon and Chrysolophus) and other 11 genera of Phasianidae in China, a total of 1070 nucleotides of mitochondrial DNA (mtDNA) control-region genes were sequenced. There are 376 variable sites including 345 parsimony sites. The genetic distance ranged from 0.067 (Chrysolophus and Phasianus) to 0.181 (Perdix and Bambusicola) among the 15 genera. Maximum likelihood method was used to construct a phylogenetic tree, which grouped all the genera into two deeply divergent clades. Perdix was shown to be a non-partridge genus. Alternatively, it appears ancestral to either partridges or pheasants. The sibling taxa of the four endemic genera were Lophophorus, Tragopan, Lophura and Phasianus, respectively. Calibrated rates of molecular evolution suggested that the divergence time between the four genera and related taxa was 4.00–5.00 million years ago, corresponding to the Pliocene. Considering their molecular phylogenetics, fossil and geographical distribution patterns, the four endemic genera might have originated in the southwestern mountains in China.     

A tool for aligning very similar DNA sequences

Results: We have produced a computer program, named sim3, thatsolves the following computational problem. Two DNA sequencesare given, where the shorter sequence is very similar to somecontiguous region of the longer sequence. Sim3 determines sucha similar region of the longer sequence, and then computes anoptimal set of single-nucleotide changes (i.e. insertions, deletionsor substitutions) that will convert the shorter sequence tothat region. Thus, the alignment scoring scheme is designedto model sequencing errors, rather than evolutionary processes.The program can align a 100 kb sequence to a 1 megabase sequencein a few seconds on a workstation, provided that there are veryfew differences between the shorter sequence and some regionin the longer sequence. The program has been used to assemblesequence data for the Genomes Division at the National Centerfor Biotechnology Information. Availability: A version of sim3 for UNIX machines can be obtainedby anonymous ftp from ncbi. nlm. nih. gov, in the pub/sim3 directory. Contact: For portable versions for Macs and PCs, contact zjing@sunset.nlm. nih. gov.     

Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences

A molecular phylogenetic analysis of the genus Phytophthora was performed, 113 isolates from 48 Phytophthora species were included in this analysis. Phylogenetic analyses were performed on regions of mitochondrial (cytochrome c oxidase subunit 1; NADH dehydrogenase subunit 1) and nuclear gene sequences (translation elongation factor 1alpha; beta-tubulin) and comparisons made to test for incongruence between the mitochondrial and nuclear data sets. The genus Phytophthora was confirmed to be monophyletic. In addition, results confirm that the classical taxonomic grouping as described by [Waterhouse (1963)] does not reflect true phylogenetic relations. Phytophthora species were redistributed into 8 clades, providing a more accurate representation of phylogenetic relationships within the genus Phytophthora. The evolution and transition of morphological, pathogenic, and reproductive traits was inferred from the cladogram generated in this study. Mating system was inferred to be a homoplasious trait, with at least eight independent transitions from homothallism to heterothallism observed.     

Update of AMmtDB: a database of multi-aligned Metazoa mitochondrial DNA sequences

The AMmtDB database (http://bighost.area.ba.cnr.it/mitochondriome) has been updated by collecting the multi-aligned sequences of Chordata and Invertebrata mitochondrial genes coding for proteins and tRNAs. Links to the multi-aligned mtDNA intraspecies variants, collected in VarMmtDB at the Mitochondriome web site, have been introduced. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. AMmtDB data selected through SRS can be viewed and managed using GeneDoc or other programs for the management of multi-aligned data depending on the user’s operative system. The multiple alignments have been produced with CLUSTALW and PILEUP programs and then carefully optimized manually.     

Update of AMmtDB: a database of multi-aligned metazoa mitochondrial DNA sequences

The AMmtDB database (http://bio-www.ba.cnr.it:8000/srs6/ ) has been updated by collecting the multi-aligned sequences of Chordata mitochondrial genes coding for proteins and tRNAs. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. AMmtDB data selected through SRS can be viewed and managed using GeneDoc or other programs for the management of multi-aligned data depending on the user's operative system. The multiple alignments have been produced with CLUSTALW and PILEUP programs and then carefully optimized manually.