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

'Walking the talk': How companies succeed in managing risk at sea

This paper draws upon research about risk and risk management conducted with the support of The Lloyd’s Register Educational Trust1. The wider research was large in scale comprising the analysis of over 2,300 questionnaires and a range of data relating to maritime incidents. In addition the study incorporated a detailed analysis of five case study companies. It is this latter element of the research which forms the basis for this paper which considers the differences in perceptions between shore-based, and ship-based, staff working for ship operators in relation to risk management. The paper explores the means of communication utilised for the transmission of data and ideas about safety and risk management both to, and from, management. It then goes on to consider why it is that despite considerable efforts to write and to talk about safety and risk management, many companies are unsuccessful in encouraging their sea-staff to believe that safety is a genuine company priority and therefore to adhere closely to company policy in relation to safety management

Exponential distributions of collective flow-event properties in viscous liquid dynamics

We study the statistics of flow events in the inherent dynamics in supercooled two- and three-dimensional binary Lennard-Jones liquids. Distributions of changes of the collective quantities energy, pressure and shear stress become exponential at low temperatures, as does that of the event "size" $S\equiv\sum {d_i}^2$. We show how the $S$-distribution controls the others, while itself following from exponential tails in the distributions of (1) single particle displacements $d$, involving a Lindemann-like length $d_L$ and (2) the number of active particles (with $d>d_L$).Comment: Accepter version (PRL

Estimating the density-scaling exponent of a monatomic liquid from its pair potential

This paper investigates two conjectures for calculating the density dependence of the density-scaling exponent of a single-component, pair-potential liquid with strong virial potential-energy correlations. The first conjecture gives an analytical expression for the density-scaling exponent directly in terms of the pair potential. The second conjecture is a refined version of this, which involves the most likely nearest-neighbor distance determined from the pair-correlation function. The two conjectures for the density-scaling exponent are tested by simulations of three systems, one of which is the standard Lennard-Jones liquid. While both expressions give qualitatively correct results, the second expression is more accurate

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

Utilization of Molecular Inversion Probes in Malaria Sequencing

While massively parallel sequencing of whole genomes shed light on many previously puzzling genetic questions, the high costs associated with this approach makes its use impractical when large cohorts need to be sequenced at high coverage. Available capture technologies reduces the sequencing costs by enriching template material for the regions of interest. However, these technologies are also prohibitively costly at high sample numbers. Capture methods utilizing molecular inversion probes (MIPs) offer a flexible alternative to enrich template material that multiplex well for thousands of samples and require minimal resources. Here, for our work in malaria, we extend the utility of MIPs, improving the capture length and efficiency. We have also dramatically decreased the capture time from 24-48 h to 1 h. Combined, these improvements allow the potential for rapid and reliable application of MIP captures in research and, importantly, clinical settings