Using Next-Gen Sequencing to Estimate Strain Diversity and Frequency within Infections

Targeted deep sequencing has rapidly transformed our ability to investigate environmental and infectious microbial diversity. Our lab is focused on applying deep sequencing to diversity in malaria infections. A key challenge in all deep sequencing work is determining true sequence differences from errors. While several amplicon deep sequencing clustering tools exist these tools can be CPU intensive and/or lack the sensitivity to detect down to a single base pair difference between sequences, which is a necessity for examining intrapopulation differences in malaria. We have therefore created a novel clustering and statistical framework to overcome these limitations. Our clustering algorithm provides a rapid initial clusters using a step-wise heuristic process collapsing low base quality differences. These initial clusters are then subject to statistical simulations again incorporating quality to assign p-values and refine the clusters. Here, we used several control data sets of known mixtures of 16s sequence from bacterial, Plasmodium sequence, and Hepatitis-C sequence to benchmark our pipeline against other tools demonstrating equal or improved sensitivity and specificity while providing improved speed often by several orders of magnitude. Our method also offers additional benefits such as comparing PCR replicates thereby further reducing error, removing chimeras, and clustering parasites across individual patients for population-based analyses. Additionally, our methods are concrete allowing the user to target a given number of differences between clusters allowing biologic questions to be better framed. Thus, given our accuracy, speed and flexibility, our new program, SeekDeep, should be broadly applicable to deep sequencing applications from microbiomes to HIV diversity

Measurements of the unsteady flow field within the stator row of a transonic axial-flow fan. 1: Measurement and analysis technique

This two-part paper presents laser anemometer measurements of the unsteady velocity field within the stator row of a transonic axial-flow fan. The objective is to provide additional insight into unsteady blade-row interactions within high speed compressors which affect stage efficiency, energy transfer, and other design considerations. Part 1 describes the measurement and analysis techniques used for resolving the unsteady flow field features. The ensemble-average and variance of the measured velocities are used to identify the rotor wake generated and unresolved unsteadiness, respectively. (Rotor wake generated unsteadiness refers to the unsteadiness generated by the rotor wake velocity deficit and the term unresolved unsteadiness refers to all remaining contributions to unsteadiness such as vortex shedding, turbulence, mass flow fluctuations, etc.). A procedure for calculating auto and cross correlations of the rotor wake generated and unresolved unsteady velocity fluctuations is described. These unsteady-velocity correlations have significance since they also result from a decomposition of the Navier-Stokes equations. This decomposition of the Navier-Stokes equations resulting in the velocity correlations used to describe the unsteady velocity field will also be outlined in this paper

Asking for Directions: The Case for Federal Courts to Use Certification Across Borders

For more than a decade, the bench, bar, and commentators have disagreed as to whether judges should look to decisions of international and foreign courts for guidance in resolving disputes that appear in U.S. courts. In 2003, Justice Scalia\u27s dissent in Lawrence v. Texas warned darkly that the majority\u27s citation to foreign and international sources was [d]angerous dicta that risked impos [ing] foreign moods, fads, or fashions on Americans. The next year, then-Attorney General Alberto Gonzales objected that [r]eliance on foreign law threatens to unmoor the court from the proper source of its authority. Members of Congress echoed those sentiments, some going so far as to threaten to impeach Justices who relied on such materials

1,2-Diiodo-4,5-dimethyl­benzene

The structure of the title compound, C8H8I2, conforms closely to the mm2 symmetry expected for the free mol­ecule and is the first reported structure of a diiodo­dimethyl­benzene. Repulsion by neighboring I atoms and the neighboring methyl groups opposite to them results in a slight elongation of the mol­ecule along the approximate twofold rotation axis that bis­ects the ring between the two I atoms. In the extended structure, the mol­ecules form inversion-related pairs which are organized in approximately hexa­gonal close-packed layers and the layers then stacked so that mol­ecules in neighboring layers abut head-to-tail in a manner that optimizes dipole–dipole inter­actions

Measurement of Synchrotron x-ray energies and line shapes using diffraction markers

Standard reference markers for accurate, reproducible synchrotron x-ray energies are obtained using a three Si crystal spectrometer. The first two crystals are in the monochromator and the third is used to obtain diffraction markers which monitor the energy. Then for any value of the glancing angle on the reference Si crystal the energy for the (333) diffraction must occur at 3/4 that of the (444) and 3/5 of that for the (555). This establishes for the first time an absolute synchrotron energy scale. Higher-order diffractions are used to determine excitation line profiles. We conclude that the use of reference diffractions is necessary to measure reproducible x-ray energies and to analyze the incident photons\u27 line profile. The detection of diffractions near the edge of measurement and near the Cu edge will provide a fast secondary standard which will allow comparison of edge data between different laboratories. The diffraction profiles will allow the proper analysis of spectral line widths

SeekDeep: single-base resolution de novo clustering for amplicon deep sequencing

PCR amplicon deep sequencing continues to transform the investigation of genetic diversity in viral, bacterial, and eukaryotic populations. In eukaryotic populations such as Plasmodium falciparum infections, it is important to discriminate sequences differing by a single nucleotide polymorphism. In bacterial populations, single-base resolution can provide improved resolution towards species and strains. Here, we introduce the SeekDeep suite built around the qluster algorithm, which is capable of accurately building de novo clusters representing true, biological local haplotypes differing by just a single base. It outperforms current software, particularly at low frequencies and at low input read depths, whether resolving single-base differences or traditional OTUs. SeekDeep is open source and works with all major sequencing technologies, making it broadly useful in a wide variety of applications of amplicon deep sequencing to extract accurate and maximal biologic information

The G-O Rule and Waldmeier Effect in the Variations of the Numbers of Large and Small Sunspot Groups

We have analysed the combined Greenwich and Solar Optical Observing Network (SOON) sunspot group data during the period of 1874-2011 and determined variations in the annual numbers (counts) of the small, large and big sunspot groups (these classifications are made on the basis of the maximum areas of the sunspot groups). We found that the amplitude of an even-numbered cycle of the number of large groups is smaller than that of its immediately following odd-numbered cycle. This is consistent with the well known Gnevyshev and Ohl rule or G-O rule of solar cycles, generally described by using the Zurich sunspot number (Rz). During cycles 12-21 the G-O rule holds good for the variation in the number of small groups also, but it is violated by cycle pair (22, 23) as in the case of Rz. This behaviour of the variations in the small groups is largely responsible for the anomalous behaviour of Rz in cycle pair (22, 23). It is also found that the amplitude of an odd-numbered cycle of the number of small groups is larger than that of its immediately following even-numbered cycle. This can be called as `reverse G-O rule'. In the case of the number of the big groups, both cycle pairs (12, 13) and (22, 23) violated the G-O rule. In many cycles the positions of the peaks of the small, large, and big groups are different and considerably differ with respect to the corresponding positions of the Rz peaks. In the case of cycle 23, the corresponding cycles of the small and large groups are largely symmetric/less asymmetric (Waldmeier effect is weak/absent) with their maxima taking place two years later than that of Rz. The corresponding cycle of the big groups is more asymmetric (strong Waldmeier effect) with its maximum epoch taking place at the same time as that of Rz.Comment: 13 pages, 5 figures, 1 table, accepted by Solar Physic

Thermally Resistant Polymers for Use as Fuel Tanks Sealants Annual Summary Report, 1 Jul. 1968 - 30 Jun. 1969

Siloxane and perfluoroalkylenic polymers for elastomeric fuel tank sealant material

Observations and a model of undertow over the inner continental shelf

Author Posting. © American Meteorological Society, 2008. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 38 (2008): 2341-2357, doi:10.1175/2008JPO3986.1.Onshore volume transport (Stokes drift) due to surface gravity waves propagating toward the beach can result in a compensating Eulerian offshore flow in the surf zone referred to as undertow. Observed offshore flows indicate that wave-driven undertow extends well offshore of the surf zone, over the inner shelves of Martha’s Vineyard, Massachusetts, and North Carolina. Theoretical estimates of the wave-driven offshore transport from linear wave theory and observed wave characteristics account for 50% or more of the observed offshore transport variance in water depths between 5 and 12 m, and reproduce the observed dependence on wave height and water depth. During weak winds, wave-driven cross-shelf velocity profiles over the inner shelf have maximum offshore flow (1–6 cm s−1) and vertical shear near the surface and weak flow and shear in the lower half of the water column. The observed offshore flow profiles do not resemble the parabolic profiles with maximum flow at middepth observed within the surf zone. Instead, the vertical structure is similar to the Stokes drift velocity profile but with the opposite direction. This vertical structure is consistent with a dynamical balance between the Coriolis force associated with the offshore flow and an along-shelf “Hasselmann wave stress” due to the influence of the earth’s rotation on surface gravity waves. The close agreement between the observed and modeled profiles provides compelling evidence for the importance of the Hasselmann wave stress in forcing oceanic flows. Summer profiles are more vertically sheared than either winter profiles or model profiles, for reasons that remain unclear.This research was funded by the Ocean Sciences Division of the National Science Foundation under Grants OCE-0241292 and OCE-0548961

Consent Is Not Enough: Why States Must Respect the Intensity Threshold in Transnational Conflict

It is widely accepted that a state cannot treat a struggle with an organized non-state actor as an armed conflict until the violence crosses a minimum threshold of intensity. For instance, during the recent standoff at the Oregon wildlife refuge, the US. government could have lawfully used force pursuant to its domestic law enforcement and human rights obligations, but President Obama could not have ordered a drone strike on the protesters. The reason for this uncontroversial rule is simple-not every riot or civil disturbance should be treated like a war. But what if President Obama had invited Canada to bomb the protestors-once the United States consented, would all bets be off? Can an intervening state use force that would be illegal for the host state to use itself? The silence on this issue is dangerous, in no small part because these once-rare conflicts are now commonplace. States are increasingly using force against organized non-state actors outside of the states\u27 own territories-usually, though not always, with the consent of the host state. What constrains the scope of the host state\u27s consent? And can the intervening state always presume that consent is valid? This Article argues that a host state\u27s authority to consent is limited and that intervening states cannot treat consent as a blank check. Accordingly, even in consent-based interventions, the logic andf oundational norms of the internationall egal order require both consent-giving and consent-receiving states to independently evaluate what legal regime governs-this will often turn on whether the intensity threshold has been met. Ifa non-international armed conflict exists, the actions of the intervening state are governed by international humanitarian law; if not, its actions are governed instead by its own and the host state\u27s human rights obligations