Targeted high-throughput sequencing of tagged nucleic acid samples

High-throughput 454 DNA sequencing technology allows much faster and more cost-effective sequencing than traditional Sanger sequencing. However, the technology imposes inherent limitations on the number of samples that can be processed in parallel. Here we introduce parallel tagged sequencing (PTS), a simple, inexpensive and flexible barcoding technique that can be used for parallel sequencing any number and type of double-stranded nucleic acid samples. We demonstrate that PTS is particularly powerful for sequencing contiguous DNA fragments such as mtDNA genomes: in theory as many as 250 mammalian mtDNA genomes can be sequenced in a single GS FLX run. PTS dramatically increases the sequencing throughput of samples in parallel and thus fully mobilizes the resources of the 454 technology for targeted sequencing.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.      

The role of pre-emptive culling in the control of foot-and-mouth disease

The 2001 foot-and-mouth disease epidemic was controlled by culling of infectious premises and pre-emptive culling intended to limit the spread of disease. Of the control strategies adopted, routine culling of farms that were contiguous to infected premises caused the most controversy. Here we perform a retrospective analysis of the culling of contiguous premises as performed in 2001 and a simulation study of the effects of this policy on reducing the number of farms affected by disease. Our simulation results support previous studies and show that a national policy of contiguous premises (CPs) culling leads to fewer farms losing livestock. The optimal national policy for controlling the 2001 epidemic is found to be the targeting of all contiguous premises, whereas for localized outbreaks in high animal density regions, more extensive fixed radius ring culling is optimal. Analysis of the 2001 data suggests that the lowest-risk CPs were generally prioritized for culling, however, even in this case, the policy is predicted to be effective. A sensitivity analysis and the development of a spatially heterogeneous policy show that the optimal culling level depends upon the basic reproductive ratio of the infection and the width of the dispersal kernel. These analyses highlight an important and probably quite general result: optimal control is highly dependent upon the distance over which the pathogen can be transmitted, the transmission rate of infection and local demography where the disease is introduced.     

Chromosome-specific recombinant DNA libraries from the fungus Aspergillus nidulans.

Development of physical genomic maps is facilitated by identification of overlapping recombinant DNA clones containing long chromosomal DNA inserts. To simplify the analysis required to determine which clones in a genomic library overlap one another, we partitioned Aspergillus nidulans cosmid libraries into chromosome-specific subcollections. The eight A. nidulans chromosomes were resolved by pulsed field gel electrophoresis and hybridized to filter replicas of cosmid libraries. The subcollections obtained appeared to be representative of the chromosomes based on the correspondence between subcollection size and chromosome length. A sufficient number of clones was obtained in each chromosome-specific subcollection to predict the overlap and assembly of individual clones into a limited number of contiguous regions. This approach should be applicable to many organisms whose genomes can be resolved by pulsed field gel electrophoresis.     

Microspatial heterogeneity in the distribution of ciliates in a small pond

Five transects of contiguous samples from the surface of a small pond and one transect from its bottom were collected in order to quantify microspatial heterogeneity in the distribution of ciliated protozoa. Examination of the frequency-abundance relations for these transects suggests that they can be approximated by negative binomial distributions with a commonk of 1.87. Contagiousness or crowding increases with population density.Mean patch size and mean interpatch distance were measured for 4 transects as 1.5 to 2 cm and 3 to 4 cm, respectively. This heterogeneity is suggested to arise from behavioral aggregation about discrete food sources and be very ephemeral.Blocking of adjacent contiguous samples was used to investigate the effect of sample size on the apparent correlation between the numbers of pairs of taxa. In all cases examined, taxa were relatively independent in their distribution at small sample sizes and became more negatively or positively associated as samples were combined. This may reflect that the small scale patches are essentially monospecific.     

Extraction of symbolic determinants common to a family of biological sequences

A set of sequences can be defined by their common subsequences, and the length of these is a measure of the overall resemblance of the set. Each subsequence corresponds to a succession of symbols embedded in every sequence, following the same order but not necessarily contiguous. Determining the longest common subsequence (LCS) requires the exhaustive testing of all possible common subsequences, which sum up to about 2L, if L is the length of the shortest sequence. We present a polynomial algorithm (O(n X L4), where n is the number of sequences) for generating strings related to the LCS and constructed with the sequence alphabet and an indetermination symbol. Such strings are iteratively improved by deleting indetermination symbols and concomitantly introducing the greatest number of alphabet symbols. Processed accordingly, nucleic acid and protein sequences lead to key-words encompassing the salient positions of homologous chains, which can be used for aligning or classifying them, as well as for finding related sequences in data banks.     

DNA bis-intercalation: Theoretical calculation of binding curves

A statistical mechanical calculation of the binding properties of DNA bis-intercalators is presented, based on the sequence-generating function method of Lifson. The effects of binding by intercalation of one or both chromophores of a bifunctional intercalating agent are examined. The secular equation for a general model that includes the effects of neighbor (nearest and non-nearest) exclusion and/or cooperativity in the binding of both singly and doubly intercalated ligands is derived. Numerical results for binding curves are presented for a more restricted model in which each type of bound ligand rigorously excludes its nearest neighbor and the total number of sites covered by a doubly intercalated ligand is variable. At low values of free ligand concentration bis-intercalation dominates the binding process, while at high value of free ligand concentration, intercalation of only one chromophore per ligand becomes significant due to the unavailability of contiguous free sites required for bis-intercalation. Also, depending on the binding parameters, the free energy of the system can be lowered by a loss of doubly intercalated ligands in favor of singly intercalated ones. Corresponding to this transition in binding mode, the average number of sites occupied by a bound ligand decreases from that characteristic of bis-intercalation to that characteristic of mono-intercalation as free ligand concentration increases. An analysis of Scatchard plots describing bis-intercalation is presented.     

POLYGONAL ASPECTS OF CELL FACES. III. CELL SIZE,CELL DIVISION,AND CELL FACE DISTRIBUTIONS

Wheeler , George E. (Brooklyn Coll., Brooklyn, New York.) Polygonal aspects of cell faces. III. Cell size, cell division, and cell face distributions. Amer. Jour. Bot. 50(8): 747–754. Illus. 1963.—The effects of cell size differences on cell face (polygon type) distributions, and the relationship of cell division to these effects were investigated, using published and original data. Only “relative” size was considered, i.e., the size of a body compared with the sizes of contiguous bodies. A preliminary study of mixtures including 2 sizes of nonliving bodies (bubbles or shot) showed that, with increase in number of smaller bodies relative to larger, 4- and 5-gons decrease and 6-gons increase on both large and small bodies (except for 5-gons on small bubbles). Since the cited papers on cells generally include no volume measurements, cell size was assumed to be proportional to number of faces; therefore, the cells from each sample were pooled in 3 groups of different ranges as to number of faces per cell. This device made evident the 4- and 5-gon decreases and the 6-gon increases with increasing size, but only in certain samples;- marked exceptions occurred among others. New data from 2 original samples showed, in general, the correlations described above, but again then; were exceptions. Discrepancies were found to arise from variable division patterns which affect face types both directly, and indirectly through division effects on cell size. It was concluded that relative size is generally less important in determining cell face distributions than are the more direct cell division events, and that size difference effects can be detected only if the other events operate minimally.     

Ultrabroadband Mid-infrared Light Absorption Based on a Multi-cavity Plasmonic Metamaterial Array

We present a broadband plasmonic metamaterial absorber in the infrared region based on localized surface plasmon polaritons (LSPPs). The unit cell of the proposed metamaterial absorber consists of a multi-cavity structure, in which absorption resonances can be tuned independently through the modification of the width and shift of metallic walls. In order to avoid the degeneration between two contiguous resonances, which dramatically reduces the bandwidth, we introduce a zigzag design rule to arrange the cavities within a compact unit. Thus, the possible number of resonances is greatly increased, enabling an ultrabroadband absorption. A broadband absorber is demonstrated with only a few-layer structure and it also has an incident-angle-insensitive feature. Our results have potential applications in photovoltaic devices, emitters, sensors, and camouflage systems.     

Freeze drying cattle blood typing reagents*

A system is described for freeze drying and storage of reagents (antisera) used in cattle blood typing tests. Reagents were freeze-dried in evacuated bottles at the desired dilution for rapid reconstitution to use in test procedures. Reagents not in current use were dried in bulk lots and stored in polyethylene film bags. All freeze drying procedures were performed with readily available commercial equipment. A computer program was developed to produce a current inventory of reagent supplies and projections of the number of samples which can be typed.     

Coupled analysis of gene expression and chromosomal location

Microarray technology can be used to assess simultaneously global changes in expression of mRNA or genomic DNA copy number among thousands of genes in different biological states. In many cases, it is desirable to determine if altered patterns of gene expression correlate with chromosomal abnormalities or assess expression of genes that are contiguous in the genome. We describe a method, differential gene locus mapping (DIGMAP), which aligns the known chromosomal location of a gene to its expression value deduced by microarray analysis. The method partitions microarray data into subsets by chromosomal location for each gene interrogated by an array. Microarray data in an individual subset can then be clustered by physical location of genes at a subchromosomal level based upon ordered alignment in genome sequence. A graphical display is generated by representing each genomic locus with a colored cell that quantitatively reflects its differential expression value. The clustered patterns can be viewed and compared based on their expression signatures as defined by differential values between control and experimental samples. In this study, DIGMAP was tested using previously published studies of breast cancer analyzed by comparative genomic hybridization (CGH) and prostate cancer gene expression profiles assessed by cDNA microarray experiments. Analysis of the breast cancer CGH data demonstrated the ability of DIGMAP to deduce gene amplifications and deletions. Application of the DIGMAP method to the prostate data revealed several carcinoma-related loci, including one at 16q13 with marked differential expression encompassing 19 known genes including 9 encoding metallothionein proteins. We conclude that DIGMAP is a powerful computational tool enabling the coupled analysis of microarray data with genome location.     

Impact of spatial disjunction within biophysical classes on plant species composition: implications for conservation planning

The use of surrogates for biodiversity is a practical tool to improve the cost effectiveness of regional conservation planning. However, there is still much uncertainty about the biological representativeness of surrogates. Using a biophysical classification system known as the Mitchell Landscapes, we compare plant species composition in contiguous versus disjunct units of nine Landscape types and hence the ability of this surrogate to capture patterns of plant species composition. We found that plant species homogeneity was higher within a contiguous Landscape than between non‐contiguous units of the same Landscape. Overall, the dissimilarity between non‐contiguous units and their contiguous counterparts was significant (P = 0.004). Biophysical classes with very high dissimilarities between non‐contiguous units of the same region may be of limited utility.     

A multichannel microcomputer-based system for continuously measuring and recording ion activities of uptake solutions during ion absorption by roots of intact plants

Abstract. A microcomputer-based system for the measurement of changes of ion activity (H⁺, NO₃⁻, K⁺) in the uptake solutions during ion absorption by roots of intact plants is described. Ion activities are measured by means of ion-specific electrodes in a multichannel programmable voltmeter (data acquisition/control unit) which is activated by means of a microcomputer. Incoming data are stored digitally on tape cassettes or floppy discs via the microcomputer. The speed of sampling and large numbers of samples which can be simultaneously measured and recorded make this an extremely versatile system which might be employed for measuring and recording any number of analogue voltage signals.     

Local and Regional Variation in Local Frequency of Multiple Coffee Pests Across a Mosaic Landscape in Coffea arabica's Native Range

Shaded coffee has been highlighted for its potential to conserve biodiversity, and thus perhaps also a diversity of natural enemies that could control pest organisms. In southwestern Ethiopia, coffee is grown in shade both in contiguous forests and in forest patches with native trees surrounded by open fields. We hypothesized that coffee grown in contiguous forests, which is the natural habitat for coffee (Coffea arabica) and its interacting organisms, would have less pest damage due to high protection by natural enemies. We surveyed pests on coffee plants in plots within contiguous forests (10 sites) and in forest patches (21 sites). In general, the variation in number of damaged or attacked leaves by individual insect or fungal pests was larger between plants than between plots, which suggests that very local conditions or processes are important. The spatial signals were generally weak. Coffee rust and coffee blotch miner tended to have lower infestation rates in accordance with our hypothesis, while fruit flies in ripe berries were more abundant in forest patches closer to contiguous forest. Based on interviews, olive baboons showed a clear dependency on contiguous forest habitat and were regarded as a problem only in contiguous forests and forest patches close to contiguous forests. In conclusion, we found no support for a generally stronger top‐down control on coffee pests in sites within, or with connectivity to, contiguous moist afromontane forests in the native range of coffee.     

Microdeletion and microduplication syndromes

The widespread use of whole genome analysis based on array comparative genomic hybridization in diagnostics and research has led to a continuously growing number of microdeletion and microduplication syndromes (MMSs) connected to certain phenotypes. These MMSs also include increasing instances in which the critical region can be reciprocally deleted or duplicated. This review catalogues the currently known MMSs and the corresponding critical regions including phenotypic consequences. Besides the pathogenic pathways leading to such rearrangements, the different detection methods and their limitations are discussed. Finally, the databases available for distinguishing between reported benign or pathogenic copy number alterations are highlighted. Overall, a review of MMSs that previously were also denoted "genomic disorders" or "contiguous gene syndromes" is given.     

Effects of fragmentation and landscape variation on tree diversity in post-logging regrowth forests of the Southern Philippines

The conservation value of forest fragments remains controversial. An extensive inventory of rainforest trees in post-logging regrowth forest in the southern Philippines provided a rare opportunity to compare stem density, species richness, diversity and biotic similarity between two types of post-logging forests: broken-canopy forest fragments and adjacent tracts of closed-canopy ‘contiguous’ forest. Tree density was much lower in the fragments, but rarefied species richness was higher. ‘Hill’ numbers, computed as the exponential of Shannon’s diversity index and the inverse of Simpson’s diversity index, indicated that fragments have higher numbers of typical and dominant species compared to contiguous forest. Beta diversity (based on species incidence) and the exponential of Shannon’s diversity index was higher in fragmented forest, indicating higher spatial species turnover than in contiguous forest samples. Lower mean values of the Chao-Jaccard index in fragmented forest compared to contiguous forest also indicated a lower probability of shared species across fragments. The high species richness of contiguous forest showed that an earlier single logging event had not caused biodiversity to be degraded leaving mostly generalist species. Fragmentation and further low-level utilisation by local farmers has also not caused acute degradation. Post-logging regrowth forest fragments present a window of opportunity for conservation that may disappear in a few years as edge effects become more apparent. For the conservation of trees in forests in south-east Asia generally, our findings also suggest that while conservation of remaining primary forest may be preferable, the conservation value of post-logging regrowth forests can also be high.     

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete -helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.