Unlocking Short Read Sequencing for Metagenomics

Background

Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved.

Methodology/Principal Findings

We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read.

Conclusions/Significance

This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.     

Origin and history of mitochondrial DNA lineages in domestic horses

Domestic horses represent a genetic paradox: although they have the greatest number of maternal lineages (mtDNA) of all domestic species, their paternal lineages are extremely homogeneous on the Y-chromosome. In order to address their huge mtDNA variation and the origin and history of maternal lineages in domestic horses, we analyzed 1961 partial d-loop sequences from 207 ancient remains and 1754 modern horses. The sample set ranged from Alaska and North East Siberia to the Iberian Peninsula and from the Late Pleistocene to modern times. We found a panmictic Late Pleistocene horse population ranging from Alaska to the Pyrenees. Later, during the Early Holocene and the Copper Age, more or less separated sub-populations are indicated for the Eurasian steppe region and Iberia. Our data suggest multiple domestications and introgressions of females especially during the Iron Age. Although all Eurasian regions contributed to the genetic pedigree of modern breeds, most haplotypes had their roots in Eastern Europe and Siberia. We found 87 ancient haplotypes (Pleistocene to Mediaeval Times); 56 of these haplotypes were also observed in domestic horses, although thus far only 39 haplotypes have been confirmed to survive in modern breeds. Thus, at least seventeen haplotypes of early domestic horses have become extinct during the last 5,500 years. It is concluded that the large diversity of mtDNA lineages is not a product of animal breeding but, in fact, represents ancestral variability.     

Pollen subsidies promote whitefly control through the numerical response of predatory mites

Several studies have shown that biological control of pests can be improved by supplying extra food to natural enemies. This increases population levels of the enemies, resulting in decreases in pest densities. In theory, however, supplying food can also have negative effects on biological control. We specifically tested for such negative effects, using a predator–prey system consisting of the whitefly Bemisia tabaci (Gennadius) and a predatory mite Amblyseius swirskii (Athias-Henriot). This predator attacks eggs and young instars of the whitefly, but also feeds on pollen. We added pollen to populations of predators and whiteflies on isolated cucumber plants. Although the set-up of our experiments would favour the occurrence of a negative effect of the addition of pollen on biological control, we found increased control throughout the experiment. This shows that the control of whiteflies by A. swirskii can be improved by supplementing the predators with pollen.     

The effect of application techniques on field‐scale efficacy: can the use of entomopathogenic nematodes reduce damage by western corn rootworm larvae?

• 1 Field studies on the efficacy of entomopathogenic nematodes as biological pest control agents in field crops and over multiple seasons are rare.
• 2 In the present study, we investigated the efficacy of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) at reducing maize root damage caused by the larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). The study consisted of 21 field‐scale experiments in Hungary conducted between 2004 and 2007. Key factors behind successful control, such as application techniques, nematode dosage, soil environment and rainfall, were analyzed.
• 3 Five of the six tested techniques for applying the nematodes using readily available farming machinery resulted in a reduction of root damage, as well as subsequent plant lodging.
• 4 According to damage rating with the Iowa 1–6 scale, damage was reduced by 3–18%. According to the 0.00–3.00 node injury scale, damage was reduced by 14–54%. Approximately 7–62 % of plant lodging, and thus direct yield losses as a result of in‐harvestability, was prevented.
• 5 An increase in nematode dosage and/or an increased rainfall during the period of applications increased the efficacy of H. bacteriophora at reducing root damage. Except for the obvious effect of moisture, the soil environment had little measurable influence on nematode efficacy.
• 6 The present study demonstrates the potential for field‐scale nematode application to significantly reduce damage caused by D. v. virgifera larvae. It is recommended that the method of agent application should be selected on a case‐by‐case basis considering the efficacy data presented here, the available application machinery and its associated costs.

     

Intensive Farming: Evolutionary Implications for Parasites and Pathogens

An increasing number of scientists have recently raised concerns about the threat posed by human intervention on the evolution of parasites and disease agents. New parasites (including pathogens) keep emerging and parasites which previously were considered to be 'under control' are re-emerging, sometimes in highly virulent forms. This re-emergence may be parasite evolution, driven by human activity, including ecological changes related to modern agricultural practices. Intensive farming creates conditions for parasite growth and transmission drastically different from what parasites experience in wild host populations and may therefore alter selection on various traits, such as life-history traits and virulence. Although recent epidemic outbreaks highlight the risks associated with intensive farming practices, most work has focused on reducing the short-term economic losses imposed by parasites, such as application of chemotherapy. Most of the research on parasite evolution has been conducted using laboratory model systems, often unrelated to economically important systems. Here, we review the possible evolutionary consequences of intensive farming by relating current knowledge of the evolution of parasite life-history and virulence with specific conditions experienced by parasites on farms. We show that intensive farming practices are likely to select for fast-growing, early-transmitted, and hence probably more virulent parasites. As an illustration, we consider the case of the fish farming industry, a branch of intensive farming which has dramatically expanded recently and present evidence that supports the idea that intensive farming conditions increase parasite virulence. We suggest that more studies should focus on the impact of intensive farming on parasite evolution in order to build currently lacking, but necessary bridges between academia and decision-makers.     

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in?vitro and in?vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.     

Cues of intraguild predators affect the distribution of intraguild prey

Theory on intraguild (IG) predation predicts that coexistence of IG-predators and IG-prey is only possible for a limited set of parameter values, suggesting that IG-predation would not be common in nature. This is in conflict with the observation that IG-predation occurs in many natural systems. One possible explanation for this difference might be antipredator behaviour of the IG-prey, resulting in decreased strength of IG-predation. We studied the distribution of an IG-prey, the predatory mite Neoseiulus cucumeris (Acari: Phytoseiidae), in response to cues of its IG-predator, the predatory mite Iphiseius degenerans. Shortly after release, the majority of IG-prey was found on the patch without cues of IG-predators, suggesting that they can rapidly assess predation risk. IG-prey also avoided patches where conspecific juveniles had been killed by IG-predators. Because it is well known that antipredator behaviour in prey is affected by the diet of the predator, we also tested whether IG-prey change their distribution in response to the food of the IG-predators (pollen or conspecific juveniles), but found no evidence for this. The IG-prey laid fewer eggs on patches with cues of IG-predators than on patches without cues. Hence, IG-prey changed their distribution and oviposition in response to cues of IG-predators. This might weaken the strength of IG-predation, possibly providing more opportunities for IG-prey and IG-predators to co-exist.     

MPRAP: An accessibility predictor for <Emphasis Type="Italic">a</Emphasis>-helical transmem-brane proteins that performs well inside and outside the membrane

Background  

In water-soluble proteins it is energetically favorable to bury hydrophobic residues and to expose polar and charged residues. In contrast to water soluble proteins, transmembrane proteins face three distinct environments; a hydrophobic lipid environment inside the membrane, a hydrophilic water environment outside the membrane and an interface region rich in phospholipid head-groups. Therefore, it is energetically favorable for transmembrane proteins to expose different types of residues in the different regions.