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Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Discussed are research procedures undertaken to determine the magnitude and timing of eustatic sea-level changes during the Tertiary Period. Data now becoming available give scientists a knowledge of conditions that may have been conducive to the formation of petroleum. (BT)

• Silvicultural management systems differ in their field-measured tree height distributions and the height distributions obtained by means of airborne laser scanning. • The forest attributes of different management systems can be successfully predicted by using joint models based on airborne laser scanning, which are indifferent in terms of treatment. • For forest inventories based on airborne laser scanning, there may not be a need to stratify forest lands by management system. In Finland, interest in continuous cover forestry (CCF) has increased rapidly in recent years. During those years CCF has been examined from various viewpoints but not from the perspective of forest inventories. This holds especially true for applications based on remote sensing. Conversely, airborne laser scanning (ALS) data have been widely used to predict forest characteristics such as size distribution and vertical forest structure, which are closely related to the forest information needs of CCF. In this study we used the area-based approach to predict a set of stand attributes from ALS data (5 pulses per m2) in a CCF forest management experiment in Katajamäki, eastern Finland. In addition to the CCF stands, the experiment included shelterwood stands and untreated stands. The predicted attributes included volume, biomass, basal area, number of stems, mean diameter, Lorey's height, dominant height, standing dead wood volume, parameters of the theoretical stem diameter distribution model, understory height and number of understory stems. Our main aim was to test whether the same model could be used across different management systems. The accuracy of the attributes predicted for the CCF stands was compared with the predictions for the other management systems in the same experiment. We also compared and discussed our results in relation to the even-aged stand attribute predictions that were conducted by using separate operational forest data collected from sites surrounding Katajamäki. The results showed that forest data from the different management systems could be combined into a single model of a stand attribute, i.e., ALS metrics were found to be suitable for comparing different management systems in regard to differences in forest structure. The accuracy of the predicted attributes in the CCF plots was comparable to that of the other management alternatives in the experiment. The accuracy was also comparable to that of even-aged forests. The results of this study were promising; the stand attributes of CCF-managed forests could be predicted analogously to those of other management systems. This indicates that for the purposes of forest inventories there may not be a need to stratify forest lands by management system. It should be noted, however, that the study area was relatively small, that the forest stands were harvested in the 1980 s, and that the attributes may not have been completely exhaustive for CCF. [ABSTRACT FROM AUTHOR]

A region of approximately one megabase of human Chromosome 12 shows extensive linkage disequilibrium in Utah residents with ancestry from northern and western Europe. This strikingly large linkage disequilibrium block was analyzed with statistical and experimental methods to determine whether natural selection could be implicated in shaping the current genome structure. Extended Haplotype Homozygosity and Relative Extended Haplotype Homozygosity analyses on this region mapped a core region of the strongest conserved haplotype to the exon 1 of the Spinocerebellar ataxia type 2 gene (SCA2). Direct DNA sequencing of this region of the SCA2 gene revealed a significant association between a pre-expanded allele \([(CAG)_8CAA(CAG)_4CAA(CAG)_8]\) of CAG repeats within exon 1 and the selected haplotype of the SCA2 gene. A significantly negative Tajima's D value (−2.20, p < 0.01) on this site consistently suggested selection on the CAG repeat. This region was also investigated in the three other populations, none of which showed signs of selection. These results suggest that a recent positive selection of the pre-expansion SCA2 CAG repeat has occurred in Utah residents with European ancestry., Organismic and Evolutionary Biology, Version of Record

• The number of living European aspen declined by 37% in just 18 years. • Aspen recruitment was severely lacking despite a large quantity of small saplings. • Recruitment was mostly inhibited by resource competition, and less by browsing. • Browsing was a stronger limiting factor nearer the forest edge. • Forest edges appear to facilitate aspen recruitment, regardless of browsing. The conservation of habitats and ecosystems is widely regarded as the most efficient way to maintain biodiversity. Protected areas are, however, under the continuous influence of natural dynamics that may affect their structure and functioning. In boreal forests, succession in protected forests may change their ecological properties, including tree species composition. European aspen (Populus tremula) hosts high species richness but appears to recruit slowly in old-growth forests. We explored the long-term dynamics of aspen in a network of forest reserves through two full-coverage inventories in 15 forest reserves in Finland, 18 years apart. We found a decline of 37% in the number of living aspen (diameter at breast height ≥ 5 cm) between the two inventories, from an average of 8 trees/ha to 5 trees/ha. The size distribution of living aspen shifted towards a greater proportion of large trees ≥ 35 cm in diameter. In addition, the rate of recruitment was low, despite a large number of saplings. Most of the observed recruitment took place along the old-growth forest edges, whereas saplings were abundantly present in all parts of the forest in conjunction with living aspen, which function as parent trees for vegetative reproduction. We also observed that browsing on aspen saplings by herbivores was greater at the forest edges and was reduced towards the forest interior. The observed decline in aspen, together with a low recruitment rate, seems to reduce the capability of these protected areas to maintain their biodiversity levels. However, and contrary to several previous suggestions, ecological processes near forest edges may mimic the natural disturbances needed to facilitate aspen recruitment. [ABSTRACT FROM AUTHOR]

• Cavities had a median persistence time of 10 years. • Persistence of a cavity depended on both tree- and stand-level factors. • Cavities persisted longest in the largest trees, except in European aspens. • Cavities persisted longer in healthy and weakened, living trees than in dead trees. • There was significant spatial variation in the risk of cavity persistence. Tree cavities are microhabitats used by multiple taxa and are considered indicators of forest biodiversity. The factors that affect cavity occurrence and its dynamics are poorly known. We studied tree- and stand-level factors that affect cavity persistence in boreal forests. Cavities of Eurasian Three-toed Woodpeckers (Picoides tridactylus) (n = 654) were surveyed in a 170 km2 area in southern Finland for 31 years during 1987–2017. In total, 447 cavities were lost during the study period: 329 to tree fall or breakage, 72 to cavity damage, and 46 to logging. With the Kaplan-Meier method and Cox proportional hazards models, we analysed which tree- and stand-level factors affected the risk of a cavity to be damaged or lost due to tree fall. The median lifespan of cavities was 10 years, and both tree- and stand-level factors affected the persistence of cavities. Cavities in managed forest areas with low territory occupancy rates were more at risk of tree fall and cavity damage than in other types of forest areas. In all nest tree species, with the exception of European aspen (Populus tremula), the risk of cavity loss to tree fall declined with increasing tree diameter. Moreover, the risk of cavity loss to tree fall was higher in dead trees than in healthy and weakened trees. Cavities in deciduous tree species other than aspen were more at risk of damage than in other tree species. Furthermore, the risk of cavity damage was greater in cavities located lower in a tree. This study showed that factors like the tree species, and size and condition of a tree modify the persistence of woodpecker-made cavities. Consequently, it is likely that these factors influence the value of cavities as microhabitats and how these cavities can be used as indicators of forest biodiversity. Understanding the dynamics of cavities in forest habitats appears critical for the use of cavities as general biodiversity indicators. [ABSTRACT FROM AUTHOR]

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.

Background: Drug metabolism is a multistep process by which the body disposes of xenobiotic agents such as therapeutic drugs. Genetic variation in the enzymes involved in this process can lead to variability in a patient's response to medication., Methods: We used molecular-inversion probe technology to develop a multiplex genotyping assay that can simultaneously test for 1227 genetic variants in 169 genes involved in drug metabolism, excretion, and transport. Within this larger set of variants, we performed analytical validation of a clinically defined core set of 165 variants in 27 genes to assess accuracy, imprecision, and dynamic range., Results: In a test set of 91 samples, genotyping accuracy for the core set probes was 99.8% for called genotypes, with a 1.2% no-call (NC) rate. The majority of the core set probes (133 of 165) had < or = 1 genotyping failure in the test set; a subset of 12 probes was responsible for the majority of failures (mainly NC). Genotyping results were reproducible upon repeat testing with overall within- and between-run variation of 1.1% and 1.4%, respectively-again, primarily NCs in a subset of probes. The assay showed stable genotyping results over a 6-fold range of input DNA., Conclusions: This assay generates a comprehensive assessment of a patient's metabolic genotype and is a tool that can provide a more thorough understanding of patient-to-patient variability in pharmacokinetic responses to drugs.

The combined effects of multiple polymorphisms in several drug-metabolizing enzyme and transporter genes can contribute to considerable interindividual variation in drug disposition and response. Therefore, it has been of increasing interest to generate scalable, flexible and cost-effective technologies for large-scale genotyping of the drug-metabolizing enzyme and transporter genes. However, the number of drug-metabolizing enzyme and transporter gene variants exceeds the capacity of current technologies to comprehensively assess multiple polymorphisms in a single, multiplexed assay. The Targeted Genotyping System (Affymetrix, CA, USA) provides a solution to this challenge, by combining molecular inversion probe technology with universal microarrays to provide a method that is capable of analyzing thousands of variants in a single reaction, while remaining relatively insensitive to cross-reactivity between reaction components. This review will focus on the Targeted Genotyping System and how this technology was adapted to enable comprehensive analysis of drug-metabolizing enzyme and transporter gene polymorphisms.

High-throughput genotyping technologies that enable large association studies are already available. Tools for genotype determination starting from raw signal intensities need to be automated, robust, and flexible to provide optimal genotype determination given the specific requirements of a study. The key metrics describing the performance of a custom genotyping study are assay conversion, call rate, and genotype accuracy. These three metrics can be traded off against each other. Using the highly multiplexed Molecular Inversion Probe technology as an example, we describe a methodology for identifying the optimal trade-off. The methodology comprises: a robust clustering algorithm and assessment of a large number of data filter sets. The clustering algorithm allows for automatic genotype determination. Many different sets of filters are then applied to the clustered data, and performance metrics resulting from each filter set are calculated. These performance metrics relate to the power of a study and provide a framework to choose the most suitable filter set to the particular study.

The main problems in drawing causal inferences from epidemiological case-control studies are confounding by unmeasured extraneous factors, selection bias and differential misclassification of exposure. In genetics the first of these, in the form of population structure, has dominated recent debate. Population structure explained part of the significant +11.2% inflation of test statistics we observed in an analysis of 6,322 nonsynonymous SNPs in 816 cases of type 1 diabetes and 877 population-based controls from Great Britain. The remainder of the inflation resulted from differential bias in genotype scoring between case and control DNA samples, which originated from two laboratories, causing false-positive associations. To avoid excluding SNPs and losing valuable information, we extended the genomic control method by applying a variable downweighting to each SNP.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of approximately 90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.

The combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) was used to determine the preferred duplex DNA binding sites of the peptide N-methylpyrrolecarboxamide antibiotic distamycin A. After 12 rounds of REPSA, several sequences were identified that bound distamycin with an apparent affinity of 2-20 nM. Among these, the highest-affinity sites averaged 10 bp in length, suggesting that these sites may be occupied by multiple, cooperatively interacting distamycin molecules. Presently, REPSA is the only combinatorial approach that allows the identification of preferred DNA targets for small molecule ligands at physiologically relevant concentrations in solution. As such, it should prove useful in the design and screening of sequence-specific DNA-binding molecules.

Mithramycin is a DNA-binding antibiotic that has been reported to selectively affect c-myc expression [Snyder, R. C. et al., (1991) Biochemistry 30, 4290-4297]. We used in vitro transcription to investigate the specificity of mithramycin action. We found that mithramycin inhibited transcription from the human c-myc P1 and P2 promoters, as well as from a minimal adenovirus-2 major late promoter, with equal efficiencies. Mithramycin also inhibited transcription elongation by creating kinetic blockades to the passage of RNA polymerase II. These data suggest that mithramycin may inhibit transcription non-specifically by affecting general processes such as transcription elongation.