Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

We previously demonstrated H2 relaxin (RLN2) facilitates castrate resistant (CR) growth of prostate cancer (CaP) cells through PI3K/Akt/β-catenin-mediated activation of the androgen receptor (AR) pathway. As inhibition of this pathway caused only ~50% reduction in CR growth, the goal of the current study was to identify additional RLN2-activated pathways that contribute to CR growth. Next-generation sequencing (NGS)-based transcriptome and gene ontology (GO) analyses comparing LNCaP stably transfected with RLN2 (LNCaP-RLN2) versus LNCaP-vector identified differential expression of genes associated with cell proliferation (12.7% of differentially expressed genes), including genes associated with the cAMP/PKA and NFκB pathways. Subsequent molecular analyses confirmed that the cAMP/PKA and NFκB pathways play a role in facilitating H2 relaxin-mediated CR growth of CaP cells. Inhibition of PKA attenuated RLN2-mediated AR activity, inhibited proliferation and caused a small but significant increase in apoptosis. Combined inhibition of the PKA and NFκB signaling pathways via inhibition of PKA and Akt induced significant apoptosis and dramatically reduced clonogenic potential, outperforming docetaxel, the standard of care treatment for CR CaP. Immunohistochemical (IHC) analysis of tissue microarrays (TMA) in combination with multispectral quantitative imaging comparing RLN2 levels in patients with BPH, PIN and CaP determined that RLN2 is significantly upregulated in CaP vs BPH (p=0.002). The combined data indicate RLN2 overexpression is frequent in CaP patients and provides a growth advantage to CaP cells. A near complete inhibition of RLN2-induced CR growth can be achieved by simultaneous blockade of both pathways.

Malaria is a mosquito-borne disease caused by the Plasmodium parasite. Of the four Plasmodium species that routinely cause human malaria, P. vivax is the most widespread species outside Africa, causing ~ 18.9 million cases in 2012. P. vivax cannot be cultured continuously in vitro, which severely hampers research in non-endemic and endemic countries alike. Consequently, whole-genome sequencing has become an effective means to interrogate the biology of the P. vivax parasite. Our comparative genomic analysis of five P. vivax reference genomes and several whole-genome sequences of the closely related monkey malaria species Plasmodium cynomolgi has revealed an extraordinary level of genetic diversity and enabled characterization of novel multi-gene families and important single-copy genes. The generation of whole-genome sequences from multiple clinical isolates is also driving forward knowledge concerning the biology and evolution of the species. Understanding the biology of P. vivax is crucial to develop potential antimalarial drugs and vaccines and to achieve the goal of eliminating malaria.

Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical, We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes.

Changes in DNA methylation, causing chromosome instability and altered gene expression, have been strongly associated with carcinogenesis. Due to the involvement of methylation in cancer, methylation profiles have been heralded as promising cancer biomarkers. Here, we present a primer design pipeline and an analysis workflow that we have developed to design and analyze custom methylation panels and detect methylation status. An automated primer design pipeline for methylation sequencing has been developed, consisting of genome conversion, primer selection, amplicon tiling, and generation of optimal amplicons. Custom methylation panels can be designed using pre-converted genomes or reference genome sequences for any other organism which can then be converted. The pipeline has the capability to create custom targeted panels specific to any methylation sites of interest. The pipeline designs Ion AmpliSeq primers to enable high multiplexing and robust amplification of low abundance or degraded DNA. Following the creation of a custom panel, a complete 3-day workflow has been developed, comprising bisulfite conversion, library construction, template preparation, sequencing and data analysis. This 3-day protocol offers manual or automated library options, low input (10-20ng DNA) and a flexible multiplexed approach with quantitative information at single base pair resolution. Sequencing is performed on the Ion GeneStudio S5 system. The bioinformatics analysis has been streamlined into a downloadable plugin performing alignment and DNA methylation calling for amplicons on both the Watson and Crick strands. To evaluate the in silico performance of the primer design pipeline for targeted bisulfite sequencing, a custom methylation panel was created using a set of 48 oncology markers from the BLUEPRINT consortium. These markers were also used for the Ion AmpliSeq Methylation Panel for Cancer Research, which was compared to the custom methylation panel to evaluate the performance of the pipeline. Key metrics from in silico design such as total number of degenerate oligos, mean amplicon length and average Tm spread for the custom methylation panel are equal to or better than Ion AmpliSeq Methylation Panel for Cancer Research. To assess the sequencing performance of the panel, two control gDNA samples were used. The expected average methylation status across all CpGs were >98% and <5% for the first sample and the second sample, respectively. The evaluation was also carried out with an equal mixture of these two samples. The wet lab testing of the custom methylation panel generated comparable results to the Ion AmpliSeq Methylation Panel for Cancer Research. The primer design pipeline and 3-day workflow provide custom design of targeted methylation panels along with quantitative analysis of relevant oncology markers from low DNA input. Citation Format: Zunping Luo, Loni Pickle, Andrew Hatch, Aren Ewing, Fiona Hyland, David Berman, Palak Patel, Mark Andersen. Custom primer design pipeline and analysis workflow for targeted methylation sequencing using NGS Ion AmpliSeq technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 158.

Viruses are infectious agents that can cause many diseases including cancers. A viral species is comprised of many strains each having a unique pathology. For example, over 170 types of HPV have been described, of which only 2 strains, HPV16 and HPV18, are implicated in 70% of cervical cancer cases. Accurate identification of viral strains is often critical and also facilitates research into their association with prognosis or response and personalized therapies. Currently, sequencing virus genes or genome requires complex procedures that involve purifying the virus particle and its genomes from the host genome. Whole genome sequencing is inefficient since viral genomes are orders of magnitude smaller than human genomes, and human DNA is orders of magnitude more abundant than viral DNA. In contrast, targeted next-generation sequencing allows focusing of sequencing and analysis to the region or species of interest using PCR based enrichment. We developed a viral design pipeline that accepts a multiple sequence alignment of the viral genomes or genomic regions of interest to identify and design primer pairs in conserved regions flanking the variable regions. The presence/absence of a set of 1 or more amplicons is used to precisely differentiate the many strains of a viral species. For whole genome sequencing, the complete viral genomic sequence is used as target to design unique and highly specific amplicons covering the whole genome for enrichment of the entire virus. All primers pass through rigorous quality checks to meet our sensitivity, specificity, and primer quality standards. We have successfully designed multi-strain Ion Ampliseq custom viral panels for HCV, HIV, CMV, Measles and Pestivirus. Some of these panels were designed with the objective of targeting a specific region on a virus genome; other panels were designed to enrich and sequence the whole virus genome. The panel primers were highly specific to the region of interest in the viral genomes and did not amplify the host genome. In conclusion, our custom Ion AmpliSeq viral panels enable the enrichment and amplification of the unique regions of interest in the viral genomes or the whole virus genome, avoiding the massive excess of the host genome. These panels allow precise identification of the virus species and strains for many applications including research, phylogenomic classification and comparative genomics. For Research Use Only. Not for use in diagnostic procedures Citation Format: Ratnesh Singh, Na Li, Zunping Luo, Anna McGeachy, Antonio F. Martinez Alcantara, Aren Ewing, Fiona C. Hyland. Custom next-generation sequencing primer designs for targeted sequencing of multi strain viral targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1207.