Rapid whole genome optical mapping of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Immune evasion and drug resistance in malaria have been linked to chromosomal recombination and gene copy number variation (CNV). These events are ideally studied using comparative genomic analyses; however in malaria these analyses are not as common or thorough as in other infectious diseases, partly due to the difficulty in sequencing and assembling complete genome drafts. Recently, whole genome optical mapping has gained wide use in support of genomic sequence assembly and comparison. Here, a rapid technique for producing whole genome optical maps of <it>Plasmodium falciparum </it>is described and the results of mapping four genomes are presented.</p> <p>Methods</p> <p>Four laboratory strains of <it>P. falciparum </it>were analysed using the Argus™ optical mapping system to produce ordered restriction fragment maps of all 14 chromosomes in each genome. <it>Plasmodium falciparum </it>DNA was isolated directly from blood culture, visualized using the Argus™ system and assembled in a manner analogous to next generation sequence assembly into maps (AssemblyViewer™, OpGen Inc.^®). Full coverage maps were generated for <it>P. falciparum </it>strains 3D7, FVO, D6 and C235. A reference <it>P. falciparum in silico </it>map was created by the digestion of the genomic sequence of <it>P. falciparum </it>with the restriction enzyme AflII, for comparisons to genomic optical maps. Maps were then compared using the MapSolver™ software.</p> <p>Results</p> <p>Genomic variation was observed among the mapped strains, as well as between the map of the reference strain and the map derived from the putative sequence of that same strain. Duplications, deletions, insertions, inversions and misassemblies of sizes ranging from 3,500 base pairs up to 78,000 base pairs were observed. Many genomic events occurred in areas of known repetitive sequence or high copy number genes, including <it>var </it>gene clusters and <it>rifin </it>complexes.</p> <p>Conclusions</p> <p>This technique for optical mapping of multiple malaria genomes allows for whole genome comparison of multiple strains and can assist in identifying genetic variation and sequence contig assembly. New protocols and technology allowed us to produce high quality contigs spanning four <it>P. falciparum </it>genomes in six weeks for less than $1,000.00 per genome. This relatively low cost and quick turnaround makes the technique valuable compared to other genomic sequencing technologies for studying genetic variation in malaria.</p

The landscape of inherited and de novo copy number variants in a plasmodium falciparum genetic cross

<p>Abstract</p> <p>Background</p> <p>Copy number is a major source of genome variation with important evolutionary implications. Consequently, it is essential to determine copy number variant (CNV) behavior, distributions and frequencies across genomes to understand their origins in both evolutionary and generational time frames. We use comparative genomic hybridization (CGH) microarray and the resolution provided by a segregating population of cloned progeny lines of the malaria parasite, <it>Plasmodium falciparum</it>, to identify and analyze the inheritance of 170 genome-wide CNVs.</p> <p>Results</p> <p>We describe CNVs in progeny clones derived from both Mendelian (i.e. inherited) and non-Mendelian mechanisms. Forty-five CNVs were present in the parent lines and segregated in the progeny population. Furthermore, extensive variation that did not conform to strict Mendelian inheritance patterns was observed. 124 CNVs were called in one or more progeny but in neither parent: we observed CNVs in more than one progeny clone that were not identified in either parent, located more frequently in the telomeric-subtelomeric regions of chromosomes and singleton <it>de novo </it>CNVs distributed evenly throughout the genome. Linkage analysis of CNVs revealed dynamic copy number fluctuations and suggested mechanisms that could have generated them. Five of 12 previously identified expression quantitative trait loci (eQTL) hotspots coincide with CNVs, demonstrating the potential for broad influence of CNV on the transcriptional program and phenotypic variation.</p> <p>Conclusions</p> <p>CNVs are a significant source of segregating and <it>de novo </it>genome variation involving hundreds of genes. Examination of progeny genome segments provides a framework to assess the extent and possible origins of CNVs. This segregating genetic system reveals the breadth, distribution and dynamics of CNVs in a surprisingly plastic parasite genome, providing a new perspective on the sources of diversity in parasite populations.</p

Fungal diversity notes 1512-1610: taxonomic and phylogenetic contributions on genera and species of fungal taxa

This article is the 14th in the Fungal Diversity Notes series, wherein we report 98 taxa distributed in two phyla, seven classes, 26 orders and 50 families which are described and illustrated. Taxa in this study were collected from Australia, Brazil, Burkina Faso, Chile, China, Cyprus, Egypt, France, French Guiana, India, Indonesia, Italy, Laos, Mexico, Russia, Sri Lanka, Thailand, and Vietnam. There are 59 new taxa, 39 new hosts and new geographical distributions with one new combination. The 59 new species comprise Angustimassarina kunmingense, Asterina lopi, Asterina brigadeirensis, Bartalinia bidenticola, Bartalinia caryotae, Buellia pruinocalcarea, Coltricia insularis, Colletotrichum flexuosum, Colletotrichum thasutense, Coniochaeta caraganae, Coniothyrium yuccicola, Dematipyriforma aquatic, Dematipyriforma globispora, Dematipyriforma nilotica, Distoseptispora bambusicola, Fulvifomes jawadhuvensis, Fulvifomes malaiyanurensis, Fulvifomes thiruvannamalaiensis, Fusarium purpurea, Gerronema atrovirens, Gerronema flavum, Gerronema keralense, Gerronema kuruvense, Grammothele taiwanensis, Hongkongmyces changchunensis, Hypoxylon inaequale, Kirschsteiniothelia acutisporum, Kirschsteiniothelia crustaceum, Kirschsteiniothelia extensum, Kirschsteiniothelia septemseptatum, Kirschsteiniothelia spatiosum, Lecanora immersocalcarea, Lepiota subthailandica, Lindgomyces guizhouensis, Marthe asmius pallidoaurantiacus, Marasmius tangerinus, Neovaginatispora mangiferae, Pararamichloridium aquisubtropicum, Pestalotiopsis piraubensis, Phacidium chinaum, Phaeoisaria goiasensis, Phaeoseptum thailandicum, Pleurothecium aquisubtropicum, Pseudocercospora vernoniae, Pyrenophora verruculosa, Rhachomyces cruralis, Rhachomyces hyperommae, Rhachomyces magrinii, Rhachomyces platyprosophi, Rhizomarasmius cunninghamietorum, Skeletocutis cangshanensis, Skeletocutis subchrysella, Sporisorium anadelphiae-leptocomae, Tetraploa dashaoensis, Tomentella exiguelata, Tomentella fuscoaraneosa, Tricholomopsis lechatii, Vaginatispora flavispora and Wetmoreana blastidiocalcarea. The new combination is Torula sundara. The 39 new records on hosts and geographical distribution comprise Apiospora guiyangensis, Aplosporella artocarpi, Ascochyta medicaginicola, Astrocystis bambusicola, Athelia rolfsii, Bambusicola bambusae, Bipolaris luttrellii, Botryosphaeria dothidea, Chlorophyllum squamulosum, Colletotrichum aeschynomenes, Colletotrichum pandanicola, Coprinopsis cinerea, Corylicola italica, Curvularia alcornii, Curvularia senegalensis, Diaporthe foeniculina, Diaporthe longicolla, Diaporthe phaseolorum, Diatrypella quercina, Fusarium brachygibbosum, Helicoma aquaticum, Lepiota metulispora, Lepiota pongduadensis, Lepiota subvenenata, Melanconiella meridionalis, Monotosporella erecta, Nodulosphaeria digitalis, Palmiascoma gregariascomum, Periconia byssoides, Periconia cortaderiae, Pleopunctum ellipsoideum, Psilocybe keralensis, Scedosporium apiospermum, Scedosporium dehoogii, Scedosporium marina, Spegazzinia deightonii, Torula fici, Wiesneriomyces laurinus and Xylaria venosula. All these taxa are supported by morphological and multigene phylogenetic analyses. This article allows the researchers to publish fungal collections which are important for future studies. An updated, accurate and timely report of fungus-host and fungus-geography is important. We also provide an updated list of fungal taxa published in the previous fungal diversity notes. In this list, erroneous taxa and synonyms are marked and corrected accordingly

High-level classification of the Fungi and a tool for evolutionary ecological analyses

High-throughput sequencing studies generate vast amounts of taxonomic data. Evolutionary ecological hypotheses of the recovered taxa and Species Hypotheses are difficult to test due to problems with alignments and the lack of a phylogenetic backbone. We propose an updated phylum-and class-level fungal classification accounting for monophyly and divergence time so that the main taxonomic ranks are more informative. Based on phylogenies and divergence time estimates, we adopt phylum rank to Aphelidiomycota, Basidiobolomycota, Calcarisporiellomycota, Glomeromycota, Entomophthoromycota, Entorrhizomycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota and Olpidiomycota. We accept nine subkingdoms to accommodate these 18 phyla. We consider the kingdom Nucleariae (phyla Nuclearida and Fonticulida) as a sister group to the Fungi. We also introduce a perl script and a newick-formatted classification backbone for assigning Species Hypotheses into a hierarchical taxonomic framework, using this or any other classification system. We provide an example of testing evolutionary ecological hypotheses based on a global soil fungal data set.Peer reviewe

Functional analysis of sirtuin genes in multiple Plasmodium falciparum strains.

Plasmodium falciparum, the causative agent of severe human malaria, employs antigenic variation to avoid host immunity. Antigenic variation is achieved by transcriptional switching amongst polymorphic var genes, enforced by epigenetic modification of chromatin. The histone-modifying 'sirtuin' enzymes PfSir2a and PfSir2b have been implicated in this process. Disparate patterns of var expression have been reported in patient isolates as well as in cultured strains. We examined var expression in three commonly used laboratory strains (3D7, NF54 and FCR-3) in parallel. NF54 parasites express significantly lower levels of var genes compared to 3D7, despite the fact that 3D7 was originally a clone of the NF54 strain. To investigate whether this was linked to the expression of sirtuins, genetic disruption of both sirtuins was attempted in all three strains. No dramatic changes in var gene expression occurred in NF54 or FCR-3 following PfSir2b disruption, contrasting with previous observations in 3D7. In 3D7, complementation of the PfSir2a genetic disruption resulted in a significant decrease in previously-elevated var gene expression levels, but with the continued expression of multiple var genes. Finally, rearranged chromosomes were observed in the 3D7 PfSir2a knockout line. Our results focus on the potential for parasite genetic background to contribute to sirtuin function in regulating virulence gene expression and suggest a potential role for sirtuins in maintaining genome integrity

Robotics and agricultural technology for landmines removal in Sri Lanka

Humanitarian demining is calamity) of third world countries. The mines are cheapest weapon, built to make horrible injuries, affecting active people, with major falls-off into economic growth The clearing is ceaseless, more expensive The disaster is notably cruel in Sri Lanka, with anti-person mines spread in the northeast region. Now. clearing is engineering duly, and the humanitarian goal comes to be technical challenge. The advanced robotics fulfills clean and reliable tasks, on condition to upgrade sophistication and cost and to loose third-world appropriateness. I he challenge is la turn local machines and awareness into effective robotic aids, willingly used by the local people, and to enhance the on-going outcomes The solution to the demining problem shall be a low-cost robotic outfit with resort to nearby available resources and competences (e.g.. drawn on from the local agricultural machinery and know-how). The paper discusses an ongoing project that aim to develop a low cost robot with intelligent remote-command abilities, not as advanced achievements, rather as cheapest productivity upgrading, assembled from standard farming devices, through the shared know-how and commitment of locally involved operators

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome, nuclear ITS regions, and several mitochondrial genes, and conducted Skmer analysis. Chloroplast genomes of all eight species were assembled using a seed-based method.According to the Bayesian phylogenomic tree constructed with the complete chloroplast genomes, the C. verum (Sri Lanka) is sister to previously sequenced C. verum (NC_035236.1, KY635878.1), C. dubium and C. rivulorum. The C. verum sample from India is sister to C. litseifolium and C. ovalifolium. According to the ITS regions studied, C. verum (Sri Lanka) is sister to C. verum (NC_035236.1), C. dubium and C. rivulorum. Cinnamomum verum (India) shares an identical ITS region with C. ovalifolium, C. litseifolium, C. citriodorum, and C. capparu-coronde. According to the Skmer analysis C. verum (Sri Lanka) is sister to C. dubium and C. rivulorum, whereas C. verum (India) is sister to C. ovalifolium, and C. litseifolium. The chloroplast gene ycf1 was identified as a chloroplast barcode for the identification of Cinnamomum species. We identified an 18 bp indel region in the ycf1 gene, that could differentiate C. verum (India) and C. verum (Sri Lanka) samples tested.</p