Linked‐read sequencing enables haplotype‐resolved resequencing at population scale

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences – including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps – are still limited by the lack of high‐quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype‐resolved genome resequencing at population scale, we investigated properties of linked‐read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high‐coverage data (46–68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25–4.6 Mb (N50) and 0.27–0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher‐quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase‐sized genomes like birds, linked‐read sequencing at moderate depth opens an affordable avenue towards haplotype‐resolved genome resequencing at population scale.     

LINE-1 hypomethylation during primary colon cancer progression

Background

Methylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage.

Methodology/Principal Findings

DNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05).

Conclusion/Significance

By using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.     

Identification of a novel anti-σ<Superscript>E</Superscript> factor in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Fine tuning expression of genes is a prerequisite for the strictly human pathogen Neisseria meningitidis to survive hostile growth conditions and establish disease. Many bacterial species respond to stress by using alternative σ factors which, in complex with RNA polymerase holoenzyme, recognize specific promoter determinants. σ^E, encoded by rpoE (NMB2144) in meningococci, is known to be essential in mounting responses to environmental challenges in many pathogens. Here we identified genes belonging to the σ^E regulon of meningococci.     

Proteome analysis of early somatic embryogenesis in Picea glauca

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.     

Plant snoRNA database

The Plant snoRNA database (http://www.scri.sari.ac.uk/plant_snoRNA/) provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species. Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants. The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown. Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes. Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues.     

Impact of agricultural practices on the Zea mays L. endophytic community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.     

PCR analysis of the distribution of unicellular cyanobacterial diazotrophs in the Arabian Sea

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N2 fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nubel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67 degrees E meridian from Victoria, Seychelles, to Muscat, Oman (0.5 degrees S to 26 degrees N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29 degrees C) oligotrophic subsurface waters between 0 and 7 degrees N, but they were also found at a station north of Oman at 26 degrees N, 56 degrees 35'E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29 degrees C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.     

Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling

Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. Interleukin 13 (IL-13) is a pleiotropic cytokine produced mainly by T cells. A substantial amount of evidence suggests that IL-13 plays a critical role in the pathogenesis of asthma. Therefore, a neutralizing anti-IL-13 monoclonal antibody could provide therapeutic benefits to asthmatic patients. To test the concept we have generated a neutralizing rat anti-mouse IL-13 monoclonal antibody, and evaluated its effects in a chronic mouse model of asthma. Chronic asthma-like response was induced in ovalbumin (OVA) sensitized mice by repeated intranasal OVA challenges. After weeks of challenge, mice developed airway hyperresponsiveness (AHR) to methacholine stimulation, severe airway inflammation, hyper mucus production, and subepithelial fibrosis. When given at the time of each intranasal OVA challenge, anti-IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and most interestingly, airway remodeling. Taken together, these results strongly suggest that a neutralizing anti-human IL-13 monoclonal antibody could be an effective therapeutic agent for asthma.     

Moth species richness,abundance and diversity in fragmented urban woodlands: implications for conservation and management strategies

Urban expansion threatens global biodiversity through the destruction of natural and semi-natural habitats and increased levels of disturbance. Whilst woodlands in urban areas may reduce the impact of urbanisation on biodiversity, they are often subject to under or over-management and consist of small, fragmented patches which may be isolated. Effective management strategies for urban woodland require an understanding of the ecology and habitat requirements of all relevant taxa. Yet, little is known of how invertebrate, and in particular moth, assemblages utilise urban woodland despite being commonly found within the urban landscape. Here we show that the abundance, species richness, and species diversity of moth assemblages found within urban woodlands are determined by woodland vegetation character, patch configuration and the surrounding landscape. In general, mature broadleaved woodlands supported the highest abundance and diversity of moths. Large compact woodlands with proportionally less edge exposed to the surrounding matrix were associated with higher moth abundance than small complex woodlands. Woodland vegetation characteristics were more important than the surrounding landscape, suggesting that management at a local scale to ensure provision of good quality habitat may be relatively more important for moth populations than improving habitat connectivity across the urban matrix. Our results show that the planting of broadleaved woodlands, retaining mature trees and minimising woodland fragmentation will be beneficial for moth assemblages.     

Wine yeast typing by MALDI-TOF MS

For the production of wine, the most important industrially used yeast species is Saccharomyces cerevisiae. Years of experience have shown that wine quality and property are significantly affected by the employed strain conducting the fermentation. Consequently, the ability of a strain level differentiation became an important requirement of modern winemaking. In our study, we showed that the differentiation by time-consuming and laborious biochemical and DNA-based methods to enable a constant beverage quality and characteristics can be replaced by matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), accompanied by the additional benefit of an application prediction. Mass fingerprints of 33 Saccharomyces strains, which are commonly used for varying wine fermentations, were generated by MALDI-TOF MS upon optimized sample preparation and instrument settings and analyzed by a cluster analysis for strain or ecotype-level differentiation. As a reference method, delta-PCR was chosen to study the genetic diversity of the employed strains. Finally, the cluster analyses of both methods were compared. It could be shown that MALDI-TOF MS, acting at proteome level, provides valuable information about the relationship between yeast strains and their application potential according to their MALDI mass fingerprint.