Quantitative evaluation of bias in barcode markers derived from complex samples

PCR products have become a major commodity used to identify organisms based on polymorphism at the DNA level. One problem arising is that unbiased identification of organisms takes as working hypothesis that when DNA is extracted from a sample, a positive signal will be obtained if universal primers are used and DNA quality is suitable for PCR. As this assumption is not always correct we used a system where large differences in PCR success have been described to identify where biases appear and maybe identify solutions. Plants can be identified with at least seven independent plastid‐located loci. These differ in their degree of PCR success and how informative they are in terms of taxonomically useful sequence polymorphisms. Here we used six common plastid loci spanning 48 plant species and performed a quantitative analysis of bias at each step of the identification process. As expected we found important differences in PCR efficiency within a single species, depending on the barcoding sequence being amplified. Quantitative PCR revealed that the Ct threshold for various plastid loci, even within a single species, could exhibit greater than 2000‐fold differences in DNA quantity after amplification. We then performed Next Generation Sequencing experiments in nine species using equal quantities of three plastid‐based primers and equally‐mixed quantities of DNA from multiple species. The result was significantly biased towards species and specific loci even when using adaptor‐specific primers. Our results caution that Next‐Generation Sequencing projects may suffer dramatic bias, arising largely during DNA amplification steps. Moreover, that amplification‐based Next Generation Sequencing technologies exhibit additional bias despite using adaptor‐specific primers, indicating that amplification success depends on the DNA fragment. As such, while qualitative analysis of unknown samples are prone to false negative results if a combination of widely‐successful amplicons are not used, quantitative results should be considered highly suspect, even if all species in the starting sample are known.This work was funded by the Comunidad Autónoma de la Región de Murcia Project “Molecular markers in coservation and management of the flora of Murcia Region”

Quantitative evaluation of bias in PCR amplification and Next Generation Sequencing derived from metabarcoding samples

Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to Next-Generation Sequencing. End-point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2000-fold differences in DNA quantity after amplification. Next Generation Sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of Q-PCR amplification.This work was funded by the Comunidad Autónoma de la Región de Murcia Project “Molecular markers in conservation and management of the flora of Murcia Region”. This work was published in Analytical and Bioanalytical Chemistry (407(7): 1841-8

Superconductivity from correlated hopping

We consider a chain described by a next-nearest-neighbor hopping combined with a nearest-neighbor spin flip. In two dimensions this three-body term arises from a mapping of the three-band Hubbard model for CuO$_2$ planes to a generalized $t-J$ model and for large O-O hopping favors resonance-valence-bond superconductivity of predominantly $d$-wave symmetry. Solving the ground state and low-energy excitations by analytical and numerical methods we find that the chain is a Luther-Emery liquid with correlation exponent $K_{\rho} = (2-n)^2/2$, where $n$ is the particle density.Comment: 10 pages, RevTeX 3.0 + 2 PostScript figs. Accepted for publication in Phys.Rev.

Signatures of chaotic tunnelling

Recent experiments with cold atoms provide a significant step toward a better understanding of tunnelling when irregular dynamics is present at the classical level. In this paper, we lay out numerical studies which shed light on the previous experiments, help to clarify the underlying physics and have the ambition to be guidelines for future experiments.Comment: 11 pages, 9 figures, submitted to Phys. Rev. E. Figures of better quality can be found at http://www.phys.univ-tours.fr/~mouchet

The anaerobic digestion of pig carcase with or without sugar beet pulp, as a novel on-farm disposal method

Anaerobic digestion was investigated as a potential method for on-farm disposal of fallen stock (pig carcases), degrading the carcase material to produce biogas and digestate. The effects of feedstock (sugar beet pulp or pig carcase material or a 50:50 mix) and organic loading rate (50 g-TS L−1 or 100 g-TS L−1), during mesophilic (35 °C) anaerobic digestion were investigated. Anaerobic digestion was achieved for all experimental treatments, however the pig carcase material at the higher organic loading rate produced the second highest methane yield (0.56 Nm3 kg-VS−1 versus a range of 0.14–0.58 Nm3 kg-VS−1 for other treatments), with the highest percentage of methane in total biogas (61.6% versus a range of 36.1–55.2% for all other treatments). Satisfactory pathogen reduction is a legislative requirement for disposal of carcase material. Pathogens were quantified throughout the anaerobic digestion process. Enterococcus faecalis concentrations decreased to negligible levels (2.8 log10 CFU g-TS−1), whilst Clostridium perfringens levels remained unaffected by treatment throughout the digestion process (5.3 ± 0.2 log10 CFU g-TS−1)

Cosmological parameter estimation using Very Small Array data out to ℓ= 1500

We estimate cosmological parameters using data obtained by the Very Small Array (VSA) in its extended configuration, in conjunction with a variety of other cosmic microwave background (CMB) data and external priors. Within the flat Λ cold dark matter (ΛCDM) model, we find that the inclusion of high-resolution data from the VSA modifies the limits on the cosmological parameters as compared to those suggested by the Wilkinson Microwave Anisotropy Probe (WMAP) alone, while still remaining compatible with their estimates. We find that Ωbh2= 0.0234+0.0012−0.0014, Ωdmh2= 0.111+0.014−0.016, h= 0.73+0.09−0.05, nS= 0.97+0.06−0.03, 1010AS= 23+7−3 and τ= 0.14+0.14−0.07 for WMAP and VSA when no external prior is included. On extending the model to include a running spectral index of density fluctuations, we find that the inclusion of VSA data leads to a negative running at a level of more than 95 per cent confidence ( nrun=−0.069 ± 0.032 ), something that is not significantly changed by the inclusion of a stringent prior on the Hubble constant. Inclusion of prior information from the 2dF galaxy redshift survey reduces the significance of the result by constraining the value of Ωm. We discuss the veracity of this result in the context of various systematic effects and also a broken spectral index model. We also constrain the fraction of neutrinos and find that fν < 0.087 at 95 per cent confidence, which corresponds to mν < 0.32 eV when all neutrino masses are equal. Finally, we consider the global best fit within a general cosmological model with 12 parameters and find consistency with other analyses available in the literature. The evidence for nrun < 0 is only marginal within this model

Chaos assisted tunnelling with cold atoms

In the context of quantum chaos, both theory and numerical analysis predict large fluctuations of the tunnelling transition probabilities when irregular dynamics is present at the classical level. We consider here the non-dissipative quantum evolution of cold atoms trapped in a time-dependent modulated periodic potential generated by two laser beams. We give some precise guidelines for the observation of chaos assisted tunnelling between invariant phase space structures paired by time-reversal symmetry.Comment: submitted to Phys. Rev. E ; 16 pages, 13 figures; figures of better quality can be found at http://www.phys.univ-tours.fr/~mouchet

Random Exchange Quantum Heisenberg Chains

The one-dimensional quantum Heisenberg model with random $\pm J$ bonds is studied for $S=\frac{1}{2}$ and $S=1$. The specific heat and the zero-field susceptibility are calculated by using high-temperature series expansions and quantum transfer matrix method. The susceptibility shows a Curie-like temperature dependence at low temperatures as well as at high temperatures. The numerical results for the specific heat suggest that there are anomalously many low-lying excitations. The qualitative nature of these excitations is discussed based on the exact diagonalization of finite size systems.Comment: 13 pages, RevTex, 12 figures available on request ([email protected]

iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophages

Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of ≤ 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-α, IL-6, IL-10, GM-CSF, RANTES and IFN-β when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells

Can forest management based on natural disturbances maintain ecological resilience?

Given the increasingly global stresses on forests, many ecologists argue that managers must maintain ecological resilience: the capacity of ecosystems to absorb disturbances without undergoing fundamental change. In this review we ask: Can the emerging paradigm of natural-disturbance-based management (NDBM) maintain ecological resilience in managed forests? Applying resilience theory requires careful articulation of the ecosystem state under consideration, the disturbances and stresses that affect the persistence of possible alternative states, and the spatial and temporal scales of management relevance. Implementing NDBM while maintaining resilience means recognizing that (i) biodiversity is important for long-term ecosystem persistence, (ii) natural disturbances play a critical role as a generator of structural and compositional heterogeneity at multiple scales, and (iii) traditional management tends to produce forests more homogeneous than those disturbed naturally and increases the likelihood of unexpected catastrophic change by constraining variation of key environmental processes. NDBM may maintain resilience if silvicultural strategies retain the structures and processes that perpetuate desired states while reducing those that enhance resilience of undesirable states. Such strategies require an understanding of harvesting impacts on slow ecosystem processes, such as seed-bank or nutrient dynamics, which in the long term can lead to ecological surprises by altering the forest's capacity to reorganize after disturbance