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

Airflows inside passenger cars and implications for airborne disease transmission

Transmission of highly infectious respiratory diseases, including SARS-CoV-2 are facilitated by the transport of tiny droplets and aerosols (harboring viruses, bacteria, etc.) that are breathed out by individuals and can remain suspended in air for extended periods of time in confined environments. A passenger car cabin represents one such situation in which there exists an elevated risk of pathogen transmission. Here we present results from numerical simulations of the potential routes of airborne transmission within a model car geometry, for a variety of ventilation configurations representing different combinations of open and closed windows. We estimate relative concentrations and residence times of a non-interacting, passive scalar -- a proxy for infectious pathogenic particles -- that are advected and diffused by the turbulent airflows inside the cabin. Our findings reveal that creating an airflow pattern that travels across the cabin, entering and existing farthest from the occupants can potentially reduce the transmission.Comment: 8 pages, 6 figures + supplementa

Magellan/M2FS Spectroscopy of Galaxy Clusters: Stellar Population Model and Application to Abell 267

We report the results of a pilot program to use the Magellan/M2FS spectrograph to survey the galactic populations and internal kinematics of galaxy clusters. For this initial study, we present spectroscopic measurements for $223$ quiescent galaxies observed along the line of sight to the galaxy cluster Abell 267 ($z\sim0.23$). We develop a Bayesian method for modeling the integrated light from each galaxy as a simple stellar population, with free parameters that specify redshift ($v_\mathrm{los}/c$) and characteristic age, metallicity ($\mathrm{[Fe/H]}$), alpha-abundance ($[\alpha/\mathrm{Fe}]$), and internal velocity dispersion ($\sigma_\mathrm{int}$) for individual galaxies. Parameter estimates derived from our 1.5-hour observation of A267 have median random errors of $\sigma_{v_\mathrm{los}}=20\ \mathrm{km\ s^{-1}}$, $\sigma_{\mathrm{Age}}=1.2\ \mathrm{Gyr}$, $\sigma_{\mathrm{[Fe/H]}}=0.11\ \mathrm{dex}$,$\sigma_{[\alpha/\mathrm{Fe}]}=0.07\ \mathrm{dex}$, and$\sigma_{\sigma_\mathrm{int}}=20\ \mathrm{km\ s^{-1}}$. In a companion paper, we use these results to model the structure and internal kinematics of A267.Comment: 16 pages, 11 figures, accepted for publication in The Astronomical Journa

Prevention of sexual transmission of Ebola in Liberia through a national semen testing and counselling programme for survivors: an analysis of Ebola virus RNA results and behavioural data

BACKGROUND: Ebola virus has been detected in semen of Ebola virus disease survivors after recovery. Liberia\u27s Men\u27s Health Screening Program (MHSP) offers Ebola virus disease survivors semen testing for Ebola virus. We present preliminary results and behavioural outcomes from the first national semen testing programme for Ebola virus. METHODS: The MHSP operates out of three locations in Liberia: Redemption Hospital in Montserrado County, Phebe Hospital in Bong County, and Tellewoyan Hospital in Lofa County. Men aged 15 years and older who had an Ebola treatment unit discharge certificate are eligible for inclusion. Participants\u27 semen samples were tested for Ebola virus RNA by real-time RT-PCR and participants received counselling on safe sexual practices. Participants graduated after receiving two consecutive negative semen tests. Counsellors collected information on sociodemographics and sexual behaviours using questionnaires administered at enrolment, follow up, and graduation visits. Because the programme is ongoing, data analysis was restricted to data obtained from July 7, 2015, to May 6, 2016. FINDINGS: As of May 6, 2016, 466 Ebola virus disease survivors had enrolled in the programme; real-time RT-PCR results were available from 429 participants. 38 participants (9%) produced at least one semen specimen that tested positive for Ebola virus RNA. Of these, 24 (63%) provided semen specimens that tested positive 12 months or longer after Ebola virus disease recovery. The longest interval between discharge from an Ebola treatment unit and collection of a positive semen sample was 565 days. Among participants who enrolled and provided specimens more than 90 days since their Ebola treatment unit discharge, men older than 40 years were more likely to have a semen sample test positive than were men aged 40 years or younger (p=0.0004). 84 (74%) of 113 participants who reported not using a condom at enrolment reported using condoms at their first follow-up visit (p \u3c 0.0001). 176 (46%) of 385 participants who reported being sexually active at enrolment reported abstinence at their follow-up visit (p \u3c 0.0001). INTERPRETATION: Duration of detection of Ebola virus RNA by real-time RT-PCR varies by individual and might be associated with age. By combining behavioural counselling and laboratory testing, the Men\u27s Health Screening Program helps male Ebola virus disease survivors understand their individual risk and take appropriate measures to protect their sexual partners. FUNDING: World Health Organization and the US Centers for Disease Control and Prevention

How Lyman Alpha Emission Depends On Galaxy Stellar Mass

In this work, we show how the stellar mass (M) of galaxies affects the 3<z<4.6 Ly-alpha equivalent width (EW) distribution. To this end, we design a sample of 629 galaxies in the M range 7.6 < logM/Msun < 10.6 from the 3D-HST/CANDELS survey. We perform spectroscopic observations of this sample using the Michigan/Magellan Fiber System, allowing us to measure Ly-alpha fluxes and use 3D-HST/CANDELS ancillary data. In order to study the Ly-alpha EW distribution dependence on M, we split the whole sample in three stellar mass bins. We find that, in all bins, the distribution is best represented by an exponential profile of the form dN(M)/dEW= A(M)exp(-EW/W0(M))/W0(M). Through a Bayesian analysis, we confirm that lower M galaxies have higher Ly-alpha EWs. We also find that the fraction A of galaxies featuring emission and the e-folding scale W0 of the distribution anti- correlate with M, recovering expressions of the forms A(M)= -0.26(.13) logM/Msun+3.01(1.2) and W0(M)= -15.6(3.5) logM/Msun +166(34). These results are crucial for proper interpretation of Ly-alpha emission trends reported in the literature that may be affected by strong M selection biases.Comment: 4 pages, 5 figure

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving \u3e 15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR

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