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April 21, 2020  |  

Whole-genome sequence of the oriental lung fluke Paragonimus westermani.

Foodborne infections caused by lung flukes of the genus Paragonimus are a significant and widespread public health problem in tropical areas. Approximately 50 Paragonimus species have been reported to infect animals and humans, but Paragonimus westermani is responsible for the bulk of human disease. Despite their medical and economic importance, no genome sequence for any Paragonimus species is available.We sequenced and assembled the genome of P. westermani, which is among the largest of the known pathogen genomes with an estimated size of 1.1 Gb. A 922.8 Mb genome assembly was generated from Illumina and Pacific Biosciences (PacBio) sequence data, covering 84% of the estimated genome size. The genome has a high proportion (45%) of repeat-derived DNA, particularly of the long interspersed element and long terminal repeat subtypes, and the expansion of these elements may explain some of the large size. We predicted 12,852 protein coding genes, showing a high level of conservation with related trematode species. The majority of proteins (80%) had homologs in the human liver fluke Opisthorchis viverrini, with an average sequence identity of 64.1%. Assembly of the P. westermani mitochondrial genome from long PacBio reads resulted in a single high-quality circularized 20.6 kb contig. The contig harbored a 6.9 kb region of non-coding repetitive DNA comprised of three distinct repeat units. Our results suggest that the region is highly polymorphic in P. westermani, possibly even within single worm isolates.The generated assembly represents the first Paragonimus genome sequence and will facilitate future molecular studies of this important, but neglected, parasite group.


April 21, 2020  |  

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

As they migrated out of Africa and into Europe and Asia, anatomically modern humans interbred with archaic hominins, such as Neanderthals and Denisovans. The result of this genetic introgression on the recipient populations has been of considerable interest, especially in cases of selection for specific archaic genetic variants. Hsieh et al. characterized adaptive structural variants and copy number variants that are likely targets of positive selection in Melanesians. Focusing on population-specific regions of the genome that carry duplicated genes and show an excess of amino acid replacements provides evidence for one of the mechanisms by which genetic novelty can arise and result in differentiation between human genomes.Science, this issue p. eaax2083INTRODUCTIONCharacterizing genetic variants underlying local adaptations in human populations is one of the central goals of evolutionary research. Most studies have focused on adaptive single-nucleotide variants that either arose as new beneficial mutations or were introduced after interbreeding with our now-extinct relatives, including Neanderthals and Denisovans. The adaptive role of copy number variants (CNVs), another well-known form of genomic variation generated through deletions or duplications that affect more base pairs in the genome, is less well understood, despite evidence that such mutations are subject to stronger selective pressures.RATIONALEThis study focuses on the discovery of introgressed and adaptive CNVs that have become enriched in specific human populations. We combine whole-genome CNV calling and population genetic inference methods to discover CNVs and then assess signals of selection after controlling for demographic history. We examine 266 publicly available modern human genomes from the Simons Genome Diversity Project and genomes of three ancient homininstextemdasha Denisovan, a Neanderthal from the Altai Mountains in Siberia, and a Neanderthal from Croatia. We apply long-read sequencing methods to sequence-resolve complex CNVs of interest specifically in the Melanesianstextemdashan Oceanian population distributed from Papua New Guinea to as far east as the islands of Fiji and known to harbor some of the greatest amounts of Neanderthal and Denisovan ancestry.RESULTSConsistent with the hypothesis of archaic introgression outside Africa, we find a significant excess of CNV sharing between modern non-African populations and archaic hominins (P = 0.039). Among Melanesians, we observe an enrichment of CNVs with potential signals of positive selection (n = 37 CNVs), of which 19 CNVs likely introgressed from archaic hominins. We show that Melanesian-stratified CNVs are significantly associated with signals of positive selection (P = 0.0323). Many map near or within genes associated with metabolism (e.g., ACOT1 and ACOT2), development and cell cycle or signaling (e.g., TNFRSF10D and CDK11A and CDK11B), or immune response (e.g., IFNLR1). We characterize two of the largest and most complex CNVs on chromosomes 16p11.2 and 8p21.3 that introgressed from Denisovans and Neanderthals, respectively, and are absent from most other human populations. At chromosome 16p11.2, we sequence-resolve a large duplication of >383 thousand base pairs (kbp) that originated from Denisovans and introgressed into the ancestral Melanesian population 60,000 to 170,000 years ago. This large duplication occurs at high frequency (>79%) in diverse Melanesian groups, shows signatures of positive selection, and maps adjacent to Homo sapienstextendashspecific duplications that predispose to rearrangements associated with autism. On chromosome 8p21.3, we identify a Melanesian haplotype that carries two CNVs, a ~6-kbp deletion, and a ~38-kbp duplication, with a Neanderthal origin and that introgressed into non-Africans 40,000 to 120,000 years ago. This CNV haplotype occurs at high frequency (44%) and shows signals consistent with a partial selective sweep in Melanesians. Using long-read sequencing genomic and transcriptomic data, we reconstruct the structure and complex evolutionary history for these two CNVs and discover previously undescribed duplicated genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that show an excess of amino acid replacements consistent with the action of positive selection.CONCLUSIONOur results suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation that is absent from current reference genomes.Large adaptive-introgressed CNVs at chromosomes 8p21.3 and 16p11.2 in Melanesians.The magnifying glasses highlight structural differences between the archaic (top) and reference (bottom) genomes. Neanderthal (red) and Denisovan (blue) haplotypes encompassing large CNVs occur at high frequencies in Melanesians (44 and 79%, respectively) but are absent (black) in all non-Melanesians. These CNVs create positively selected genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that are absent from the reference genome.Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.


April 21, 2020  |  

The comparative genomics and complex population history of Papio baboons.

Recent studies suggest that closely related species can accumulate substantial genetic and phenotypic differences despite ongoing gene flow, thus challenging traditional ideas regarding the genetics of speciation. Baboons (genus Papio) are Old World monkeys consisting of six readily distinguishable species. Baboon species hybridize in the wild, and prior data imply a complex history of differentiation and introgression. We produced a reference genome assembly for the olive baboon (Papio anubis) and whole-genome sequence data for all six extant species. We document multiple episodes of admixture and introgression during the radiation of Papio baboons, thus demonstrating their value as a model of complex evolutionary divergence, hybridization, and reticulation. These results help inform our understanding of similar cases, including modern humans, Neanderthals, Denisovans, and other ancient hominins.


April 21, 2020  |  

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas, Mycoplasma and by sequences assigned to the order Clostridiales. Applying Sanger sequencing on the full-length bacterial 16S rRNA gene from the pool of 46 isolates obtained in this study showed a clear assignment of the PacBio full-length bacterial 16S rRNA gene sequences down to the genus level. One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions. Comparisons of sequence assignments between full-length and in silico derived variable 16S rRNA gene regions showed different microbial profiles with variable effects between phylogenetic groups and taxonomic ranks. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Aquella oligotrophica gen. nov. sp. nov.: A new member of the family Neisseriaceae isolated from laboratory tap water.

A bacterial strain designated as P08T was isolated from laboratory tap water during a water quality assessment in University of Malaya, Malaysia. The strain was a Gram-negative, rod-shaped, nonmotile, and aerobic bacterium. Complete genome of P08T comprised of a 2,820,660 bp chromosome with a G + C content of 36.43%. Both 16S rRNA phylogeny and phylogenetic tree inferred from the core gene matrix demonstrated that P08T formed a hitherto unknown subline within the family Neisseriaceae. Ortho average nucleotide identity (OrthoANI) values and the percentage of conserved proteins (POCP) calculated from complete genome sequence indicated low relatedness between P08T and its phylogenetic neighbors. Respiratory quinone analysis revealed Q-8 as the only detectable quinone. The predominant cellular fatty acids were identified as C14:0 , iso-C15:0 , and summed feature 3 (C16:1 ?7c/C16:1 ?6c). The polar lipids consisted of uncharacterized aminolipid, phosphatidylglycerol, and phosphatidylethanolamine. All aspects of phenotypic and phylogenetic data suggested that strain P08T represents a novel genus within family Neisseriaceae, for which the name Aquella gen. nov. is proposed. The type species of the genus is Aquella oligotrophica sp. nov., and the type strain is P08T (=LMG 29629T =DSM 100970T ). © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Whole genome sequencing of a novel, dichloromethane-fermenting Peptococcaceae from an enrichment culture

Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CHt2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, “DCMF”, was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTtextsuperscriptTM sequencing of DCMF allowed textitde novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the textitPeptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria and includes 96 predicted methylamine methyltransferases, which may provide clues to the basis of its DCM metabolism. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, available at http://www.slimsuite.unsw.edu.au/research/dcmf/.


April 21, 2020  |  

Long-read amplicon denoising.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called ‘amplicon denoising’, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.


April 21, 2020  |  

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.


April 21, 2020  |  

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.


April 21, 2020  |  

Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.


April 21, 2020  |  

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease.


April 21, 2020  |  

Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus.


April 21, 2020  |  

Chromulinavorax destructans, a pathogen of microzooplankton that provides a window into the enigmatic candidate phylum Dependentiae.

Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.


April 21, 2020  |  

One Health Genomic Surveillance of Escherichia coli Demonstrates Distinct Lineages and Mobile Genetic Elements in Isolates from Humans versus Livestock.

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli isolates (including 155 extended-spectrum ß-lactamase [ESBL]-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1,517 E. coli bacteria associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, suggesting that E. coli causing serious human infection had not directly originated from livestock. In contrast, we observed highly related isolates from the same animal species on different farms. Screening all 1,948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions in human and livestock isolates. Overall, we identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that within the confines of our sampling framework, there was limited evidence that antimicrobial-resistant pathogens associated with serious human infection had originated from livestock in our region. IMPORTANCE The increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1,517 patients with bloodstream infections were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was limited overlap in the mobile elements carrying these genes. Within the limitations of sampling, our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock in our region. Copyright © 2019 Ludden et al.


April 21, 2020  |  

Comparative analysis of the complete chloroplast genomes of seven Populus species: Insights into alternative female parents of Populus tomentosa.

Populus tomentosa, of section Populus, is distributed mainly in northern China. This species has high resistance to many diseases and insects, and it plays key roles in shelterbelts and urban afforestation in northern China. It has long been suspected to be a hybrid, but its parents remain unknown. In the present study, we report four newly sequenced complete cp genomes from section Populus and comparative genomic analyses of these new sequences and three published cp genome sequences. The seven cp genomes ranged from 155,853 bp (P. tremula var. davidiana) to 156,746 bp (P. adenopoda) in length, and their gene orders, gene numbers and GC contents were similar. We analyzed SNPs, indels, SSRs and repeats among the seven cp genomes, and eight small inversions were detected in the ndhC-trnV, rbcL-accD, petA-psbJ, trnW-trnP, rpl16-rps3, trnL-ycf15, ycf15-trnL, and ndhF-trnL intergenic regions. Furthermore, seven divergent regions (trnH-psbA, matK, psbM-psbD, ndhC-trnV, ycf1, ndhF-ccsA and ccsA-ndhD) were found in more highly variable regions. The phylogenetic tree reveals that P. tomentosa is closely related to P. alba and P. alba var. pyramidalis. Hence, P. alba was involved in the formation of P. tomentosa.


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