22,524 research outputs found
De Novo Co-Assembly Of Bacterial Genomes From Multiple Single Cells
Recent progress in DNA amplication techniques, particularly multiple displacement amplication (MDA), has made it possible to sequence and assemble bacterial genomes from a single cell. However, the quality of single cell genome assembly has not yet reached the quality of normal multicell genome assembly due to the coverage bias and errors caused by MDA. Using a template of more than one cell for MDA or combining separate MDA products has been shown to improve the result of genome assembly from few single cells, but providing identical single cells, as a necessary step for these approaches, is a challenge. As a solution to this problem, we give an algorithm for de novo co-assembly of bacterial genomes from multiple single cells. Our novel method not only detects the outlier cells in a pool, it also identies and eliminates their genomic sequences from the nal assembly. Our proposed co-assembly algorithm is based on colored de Bruijn graph which has been recently proposed for de novo structural variation detection. Our results show that de novo co-assembly of bacterial genomes from multiple single cells outperforms single cell assembly of each individual one in all standard metrics. Moreover, our de novo co-assembly also outperforms the mixed assembly in which the input datasets are simply concatenated. We implemented our algorithm in a software tool called HyDA which is available from http://chitsazlab.org/software/hyda
Ensemble Analysis of Adaptive Compressed Genome Sequencing Strategies
Acquiring genomes at single-cell resolution has many applications such as in
the study of microbiota. However, deep sequencing and assembly of all of
millions of cells in a sample is prohibitively costly. A property that can come
to rescue is that deep sequencing of every cell should not be necessary to
capture all distinct genomes, as the majority of cells are biological
replicates. Biologically important samples are often sparse in that sense. In
this paper, we propose an adaptive compressed method, also known as distilled
sensing, to capture all distinct genomes in a sparse microbial community with
reduced sequencing effort. As opposed to group testing in which the number of
distinct events is often constant and sparsity is equivalent to rarity of an
event, sparsity in our case means scarcity of distinct events in comparison to
the data size. Previously, we introduced the problem and proposed a distilled
sensing solution based on the breadth first search strategy. We simulated the
whole process which constrained our ability to study the behavior of the
algorithm for the entire ensemble due to its computational intensity. In this
paper, we modify our previous breadth first search strategy and introduce the
depth first search strategy. Instead of simulating the entire process, which is
intractable for a large number of experiments, we provide a dynamic programming
algorithm to analyze the behavior of the method for the entire ensemble. The
ensemble analysis algorithm recursively calculates the probability of capturing
every distinct genome and also the expected total sequenced nucleotides for a
given population profile. Our results suggest that the expected total sequenced
nucleotides grows proportional to of the number of cells and
proportional linearly with the number of distinct genomes
A Reference-Free Algorithm for Computational Normalization of Shotgun Sequencing Data
Deep shotgun sequencing and analysis of genomes, transcriptomes, amplified
single-cell genomes, and metagenomes has enabled investigation of a wide range
of organisms and ecosystems. However, sampling variation in short-read data
sets and high sequencing error rates of modern sequencers present many new
computational challenges in data interpretation. These challenges have led to
the development of new classes of mapping tools and {\em de novo} assemblers.
These algorithms are challenged by the continued improvement in sequencing
throughput. We here describe digital normalization, a single-pass computational
algorithm that systematizes coverage in shotgun sequencing data sets, thereby
decreasing sampling variation, discarding redundant data, and removing the
majority of errors. Digital normalization substantially reduces the size of
shotgun data sets and decreases the memory and time requirements for {\em de
novo} sequence assembly, all without significantly impacting content of the
generated contigs. We apply digital normalization to the assembly of microbial
genomic data, amplified single-cell genomic data, and transcriptomic data. Our
implementation is freely available for use and modification
Recovering complete and draft population genomes from metagenome datasets.
Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem of chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution
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Evolution of host support for two ancient bacterial symbionts with differentially degraded genomes in a leafhopper host.
Plant sap-feeding insects (Hemiptera) rely on bacterial symbionts for nutrition absent in their diets. These bacteria experience extreme genome reduction and require genetic resources from their hosts, particularly for basic cellular processes other than nutrition synthesis. The host-derived mechanisms that complete these processes have remained poorly understood. It is also unclear how hosts meet the distinct needs of multiple bacterial partners with differentially degraded genomes. To address these questions, we investigated the cell-specific gene-expression patterns in the symbiotic organs of the aster leafhopper (ALF), Macrosteles quadrilineatus (Cicadellidae). ALF harbors two intracellular symbionts that have two of the smallest known bacterial genomes: Nasuia (112 kb) and Sulcia (190 kb). Symbionts are segregated into distinct host cell types (bacteriocytes) and vary widely in their basic cellular capabilities. ALF differentially expresses thousands of genes between the bacteriocyte types to meet the functional needs of each symbiont, including the provisioning of metabolites and support of cellular processes. For example, the host highly expresses genes in the bacteriocytes that likely complement gene losses in nucleic acid synthesis, DNA repair mechanisms, transcription, and translation. Such genes are required to function in the bacterial cytosol. Many host genes comprising these support mechanisms are derived from the evolution of novel functional traits via horizontally transferred genes, reassigned mitochondrial support genes, and gene duplications with bacteriocyte-specific expression. Comparison across other hemipteran lineages reveals that hosts generally support the incomplete symbiont cellular processes, but the origins of these support mechanisms are generally specific to the host-symbiont system
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Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.
As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing
Host-linked soil viral ecology along a permafrost thaw gradient
Climate change threatens to release abundant carbon that is sequestered at high latitudes, but the constraints on microbial metabolisms that mediate the release of methane and carbon dioxide are poorly understood1,2,3,4,5,6,7. The role of viruses, which are known to affect microbial dynamics, metabolism and biogeochemistry in the oceans8,9,10, remains largely unexplored in soil. Here, we aimed to investigate how viruses influence microbial ecology and carbon metabolism in peatland soils along a permafrost thaw gradient in Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, 58% of which were detected in metatranscriptomes and presumed to be active. In silico predictions linked 35% of the viruses to microbial host populations, highlighting likely viral predators of key carbon-cycling microorganisms, including methanogens and methanotrophs. Lineage-specific virus/host ratios varied, suggesting that viral infection dynamics may differentially impact microbial responses to a changing climate. Virus-encoded glycoside hydrolases, including an endomannanase with confirmed functional activity, indicated that viruses influence complex carbon degradation and that viral abundances were significant predictors of methane dynamics. These findings suggest that viruses may impact ecosystem function in climate-critical, terrestrial habitats and identify multiple potential viral contributions to soil carbon cycling
Comparative genome analysis of Wolbachia strain wAu
BACKGROUND:
Wolbachia intracellular bacteria can manipulate the reproduction of their arthropod hosts, including inducing sterility between populations known as cytoplasmic incompatibility (CI). Certain strains have been identified that are unable to induce or rescue CI, including wAu from Drosophila. Genome sequencing and comparison with CI-inducing related strain wMel was undertaken in order to better understand the molecular basis of the phenotype.
RESULTS:
Although the genomes were broadly similar, several rearrangements were identified, particularly in the prophage regions. Many orthologous genes contained single nucleotide polymorphisms (SNPs) between the two strains, but a subset containing major differences that would likely cause inactivation in wAu were identified, including the absence of the wMel ortholog of a gene recently identified as a CI candidate in a proteomic study. The comparative analyses also focused on a family of transcriptional regulator genes implicated in CI in previous work, and revealed numerous differences between the strains, including those that would have major effects on predicted function.
CONCLUSIONS:
The study provides support for existing candidates and novel genes that may be involved in CI, and provides a basis for further functional studies to examine the molecular basis of the phenotype
The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.
Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination
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