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Sunday, September 22, 2019

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including…

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Sunday, September 22, 2019

Direct chromosome-length haplotyping by single-cell sequencing.

Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid genomes along the entire length of all chromosomes. We demonstrate this by building a complete haplotype for a HapMap individual (NA12878) at high accuracy (concordance 99.3%), without using generational information or statistical inference. By use of this approach, we mapped all meiotic recombination events in a family trio with high resolution (median range ~14 kb) and phased…

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Sunday, September 22, 2019

Metagenomic approaches to assess bacteriophages in various environmental niches.

Bacteriophages are ubiquitous and numerous parasites of bacteria and play a critical evolutionary role in virtually every ecosystem, yet our understanding of the extent of the diversity and role of phages remains inadequate for many ecological niches, particularly in cases in which the host is unculturable. During the past 15 years, the emergence of the field of viral metagenomics has drastically enhanced our ability to analyse the so-called viral ‘dark matter’ of the biosphere. Here, we review the evolution of viral metagenomic methodologies, as well as providing an overview of some of the most significant applications and findings in this…

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Sunday, September 22, 2019

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia.

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution…

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Sunday, September 22, 2019

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation…

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Sunday, September 22, 2019

Single-cell multiomics: multiple measurements from single cells.

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cell’s phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies. Copyright © 2016.…

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Sunday, September 22, 2019

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to…

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Sunday, September 22, 2019

Cytogenomic analysis of several repetitive DNA elements in turbot (Scophthalmus maximus).

Repetitive DNA plays a fundamental role in the organization, size and evolution of eukaryotic genomes. The sequencing of the turbot revealed a small and compact genome, as in all flatfish studied to date. The assembly of repetitive regions is still incomplete because it is difficult to correctly identify their position, number and array. The combination of classical cytogenetic techniques along with high quality sequencing is essential to increase the knowledge of the structure and composition of these sequences and, thus, of the structure and function of the whole genome. In this work, the in silico analysis of H1 histone, 5S…

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Sunday, September 22, 2019

The DNA methylome of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we…

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Sunday, September 22, 2019

Culture-facilitated comparative genomics of the facultative symbiont Hamiltonella defensa.

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids…

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Sunday, September 22, 2019

Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite Plasmodium praefalciparum. Little is known about the other gorilla- and chimpanzee-infecting species in the same (Laverania) subgenus as P. falciparum, but none of them are capable of establishing repeated infection and transmission in humans. To elucidate underlying mechanisms and the evolutionary history of this subgenus, we have generated multiple genomes from all known Laverania species. The completeness of our dataset allows us to conclude that interspecific gene transfers, as well as convergent evolution, were important in the evolution of these species. Striking…

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Sunday, September 22, 2019

A molecular window into the biology and epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study…

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Sunday, September 22, 2019

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d’Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences.…

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Sunday, September 22, 2019

B chromosomes of the Asian seabass (Lates calcarifer) contribute to genome variations at the level of individuals and populations.

The Asian seabass (Lates calcarifer) is a bony fish from the Latidae family, which is widely distributed in the tropical Indo-West Pacific region. The karyotype of the Asian seabass contains 24 pairs of A chromosomes and a variable number of AT- and GC-rich B chromosomes (Bchrs or Bs). Dot-like shaped and nucleolus-associated AT-rich Bs were microdissected and sequenced earlier. Here we analyzed DNA fragments from Bs to determine their repeat and gene contents using the Asian seabass genome as a reference. Fragments of 75 genes, including an 18S rRNA gene, were found in the Bs; repeats represented 2% of the…

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Sunday, September 22, 2019

Correcting palindromes in long reads after whole-genome amplification.

Next-generation sequencing requires sufficient DNA to be available. If limited, whole-genome amplification is applied to generate additional amounts of DNA. Such amplification often results in many chimeric DNA fragments, in particular artificial palindromic sequences, which limit the usefulness of long sequencing reads.Here, we present Pacasus, a tool for correcting such errors. Two datasets show that it markedly improves read mapping and de novo assembly, yielding results similar to these that would be obtained with non-amplified DNA.With Pacasus long-read technologies become available for sequencing targets with very small amounts of DNA, such as single cells or even single chromosomes.

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