Strobe sequence design for haplotype assembly

Abstract Background Humans are diploid, carrying two copies of each chromosome, one from each parent. Separating the paternal and maternal chromosomes is an important component of genetic analyses such as determining genetic association, inferring evolutionary scenarios, computing recombination rates, and detecting cis-regulatory events. As the pair of chromosomes are mostly identical to each other, linking together of alleles at heterozygous sites is sufficient to phase, or separate the two chromosomes. In Haplotype Assembly, the linking is done by sequenced fragments that overlap two heterozygous sites. While there has been a lot of research on correcting errors to achieve accurate haplotypes via assembly, relatively little work has been done on designing sequencing experiments to get long haplotypes. Here, we describe the different design parameters that can be adjusted with next generation and upcoming sequencing technologies, and study the impact of design choice on the length of the haplotype. Results We show that a number of parameters influence haplotype length, with the most significant one being the advance length (distance between two fragments of a clone). Given technologies like strobe sequencing that allow for large variations in advance lengths, we design and implement a simulated annealing algorithm to sample a large space of distributions over advance-lengths. Extensive simulations on individual genomic sequences suggest that a non-trivial distribution over advance lengths results a 1-2 order of magnitude improvement in median haplotype length. Conclusions Our results suggest that haplotyping of large, biologically important genomic regions is feasible with current technologies

Volatile Anesthetics Influence Blood-Brain Barrier Integrity by Modulation of Tight Junction Protein Expression in Traumatic Brain Injury

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema

Using a Tri-Isotope (13C, 15N, 33P) Labelling Method to Quantify Rhizodeposition

Belowground (BG) plant resource allocation, including roots and rhizodeposition, is a major source of soil organic matter. Knowledge on the amounts and turnover of BG carbon (C), nitrogen (N), and phosphorus (P) in soil is critical to the understanding of how these elements cycle in soil-plant system. However, the assumptions underlying the quantification and tracking of rhizodeposition using isotope labeling methods have hardly been tested. The main objectives of this chapter were to (i) review the different plant labeling techniques for each of the three elements; (ii) describe a novel method for the simultaneous investigation of C, N, and P rhizodeposition in sand; and (iii) test the methodological assumptions underlying quantification of rhizodeposition. Stable 13C and 15N isotopes were widely used to study rhizodeposition of plants either separately or in combination, while P radioisotopes (32P, 33P) were used to investigate root distribution. The combination of the 13CO2 single-pulse labeling with the simultaneous 15N and 33P cotton-wick stem feeding effectively labeled Canavalia brasiliensis roots and facilitated the estimation of rhizodeposited C, N, and P input from root systems. However, the isotope distribution was uneven within the root system for all three elements. Additionally, we observed a progressive translocation from shoot to roots for 15N and 33P over 15 days after labeling, while the 13C tracer was diluted with newly assimilated non-enriched C compounds over time. Younger root sections also showed higher specific activities (33P/31P) than the older ones. The relatively high 33P radioactivity recovered in sand right away at the first sampling was attributed to an artifact generated by the stem feeding labeling method. Overall, our results suggest that the assumptions underlying the use of isotope methods for studying rhizodeposition are violated, which will affect the extent of quantification of rhizodeposition. The consequences of nonhomogeneous labeling of root segments of different age require further investigation. The use of a time-integrated isotopic composition of the root is recommended to not only account for temporal variation of isotopes but also to improve the method of quantifying plant rhizodeposition