A suite of computational tools to interrogate sequence data with local haplotype analysis within complex ​Plasmodium​ infections and other microbial mixtures

The rapid development of DNA sequencing technologies has opened up new avenues of research, including the investigation of population structure within infectious diseases (both within patient and between populations). In order to take advantage of these advances in technologies and the generation of new types of data, novel bioinformatics tools are needed that won’t succumb to artifacts introduced by the data generation, and thus provide accurate and precise results. To achieve this goal I have create several tools. First, SeekDeep, a pipeline for analyzing targeted amplicon sequencing datasets from various technologies, is able to achieve 1-base resolution even at low frequencies and read depths allowing for accurate comparison between samples and the detection of important SNPs. Next, PathWeaver, a local haplotype assembler designed for complex infections and highly variable genomic regions with poor reference mapping. PathWeaver is able to create highly accurate haplotypes without generating chimeric assemblies. PathWeaver was used on the key protein in pregnancy-associated malaria Plasmodium falciparum VAR2CSA which revealed population sub-structuring within the key binding domain of the protein observed to be present globally along with confirming copy number variation. Finally, the program Carmen is able to utilize PathWeaver to augment the results from targeted amplicon approaches by reporting where and when local haplotypes have been found previously. These rigorously tested tools allow the analysis of local haplotype data from various technologies and approaches to provide accurate, precise and easily accessible results

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

Deep gene sequence cluster analyses of multi-virus infected mucosal tissue reveal enhanced transmission of acute HIV-1

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multi-virus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue while Envacute viruses penetrated the human tissue and established infection of CD4(+) T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggests that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g. acute Env HIV-1) can escape this trapped replication for systemic infection. Importance: During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa and leading to systemic infection of a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance to understanding dynamics of infections and to now design focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early as compared to chronic infection can more efficiently traverse the mucosal epithelium and transmitted to T cells, suggesting higher transmission fitness. These studies focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap

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

Emergence and evolution of Plasmodium falciparum histidine-rich protein 2 and 3 deletion mutant parasites in Ethiopia [preprint]

Malaria diagnostic testing in Africa is threatened by Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes. Among 12,572 subjects enrolled along Ethiopia’s borders with Eritrea, Sudan, and South Sudan and using multiple assays, we estimate HRP2-based rapid diagnostic tests would miss 9.7% (95% CI 8.5-11.1) of falciparum malaria cases due to pfhrp2 deletion. Established and novel genomic tools reveal distinct subtelomeric deletion patterns, well-established pfhrp3 deletions, and recent expansion of pfhrp2 deletion. Current diagnostic strategies need to be urgently reconsidered in Ethiopia, and expanded surveillance is needed throughout the Horn of Africa

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

Drug-Resistance and Population Structure of Plasmodium falciparum Across the Democratic Republic of Congo Using High-Throughput Molecular Inversion Probes

A better understanding of the drivers of the spread of malaria parasites and drug resistance across space and time is needed. These drivers can be elucidated using genetic tools. Here, a novel molecular inversion probe (MIP) panel targeting all major drug-resistance mutations and a set of microsatellites was used to genotype Plasmodium falciparum infections of 552 children from the 2013-2014 Demographic and Health Survey conducted in the Democratic Republic of the Congo (DRC). Microsatellite-based analysis of population structure suggests that parasites within the DRC form a homogeneous population. In contrast, sulfadoxine-resistance markers in dihydropteroate synthase show marked spatial structure with ongoing spread of double and triple mutants compared with 2007. These findings suggest that parasites in the DRC remain panmictic despite rapidly spreading antimalarial-resistance mutations. Moreover, highly multiplexed targeted sequencing using MIPs emerges as a cost-effective method for elucidating pathogen genetics in complex infections in large cohorts

Longitudinal Pooled Deep Sequencing of the Plasmodium vivax K12 Kelch Gene in Cambodia Reveals a Lack of Selection by Artemisinin

The emergence of artemisinin resistance among Plasmodium falciparum in the Greater Mekong subregion threatens malaria control interventions and is associated with multiple unique mutations in K13 (PF3D7_1343700). The aim of this study was to survey Cambodian Plasmodium vivax for mutations in the K13 ortholog (K12, PVX_083080) that might similarly confer artemisinin resistance. Extracted DNA from Cambodian isolates collected between 2009 and 2012 was pooled by province and year and submitted for next-generation sequencing. Single-nucleotide polymorphisms (SNPs) were identified using a pile-up approach that detected minority SNPs. Among the 14 pools, we found six unique SNPs, including three nonsynonymous SNPs, across six codons in K12. However, none of the SNPs were orthologous to artemisinin resistance–conferring mutations in PF3D7_1343700, and nonsynonymous changes did not persist through time within populations. These results suggest a lack of selection in the P. vivax population in Cambodia due to artemisinin drug pressure

Rhombohedral magnetostriction in dilute iron (Co) alloys

Iron is a well-utilized material in structural and magnetic applications. This does not mean, however, that it is well understood, especially in the field of magnetostriction. In particular, the rhombohedral magnetostriction of iron, λ111 , is anomalous in two respects: it is negative in sign, in disagreement with the prediction of first principles theory, and its magnitude decreases with increasing temperature much too rapidly to be explained by a power law dependence on magnetization. These behaviors could arise from the location of the Fermi level, which leaves a small region of the majority 3d t2g states unfilled, possibly favoring small internal displacements that split these states. If this view is correct, adding small amounts of Co to Fe fills some of these states, and the value of λ111 should increase toward a positive value, as predicted for perfect bcc Fe. We have measured the magnetostriction coefficients (λ111 and λ100) of pure Fe, Fe97Co3, and Fe94Co6 single crystals from 77 K to 450 K. Resonant ultrasound spectroscopy has been used to check for anomalies in the associated elastic constants, c 44 and c′. The additional electrons provided by the cobalt atoms indeed produced positive contributions to bothmagnetostriction constants, λ111 and λ100, exhibiting an increase of 2.8 × 10−6 per at. % Co for λ111 and 3.8 × 10−6 per at. % Co for λ100