Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum.

The "dark matter of life" describes microbes and even entire divisions of bacterial phyla that have evaded cultivation and have yet to be sequenced. We present a genome from the globally distributed but elusive candidate phylum TM6 and uncover its metabolic potential. TM6 was detected in a biofilm from a sink drain within a hospital restroom by analyzing cells using a highly automated single-cell genomics platform.

Assembly of large, high G+C bacterial DNA fragments in yeast.

The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%.

Direct transfer of whole genomes from bacteria to yeast.

Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion.

Sequencing of isolated sperm cells for direct haplotyping of a human genome.

There is increasing evidence that the phenotypic effects of genomic sequence variants are best understood in terms of variant haplotypes rather than as isolated polymorphisms. Haplotype analysis is also critically important for uncovering population histories and for the study of evolutionary genetics. Although the sequencing of individual human genomes to reveal personal collections of sequence variants is now well established, there has been slower progress in the phasing of these variants into pairs of haplotypes along each pair of chromosomes.

Metagenomic exploration of viruses throughout the Indian Ocean.

The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far.

Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast.

Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.

Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic.

Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of ~5.8 million predicted proteins across seven sites, from three different size classes: 0.

Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage.

Bacteria in the 16S rRNA clade SAR86 are among the most abundant uncultivated constituents of microbial assemblages in the surface ocean for which little genomic information is currently available. Bioinformatic techniques were used to assemble two nearly complete genomes from marine metagenomes and single-cell sequencing provided two more partial genomes. Recruitment of metagenomic data shows that these SAR86 genomes substantially increase our knowledge of non-photosynthetic bacteria in the surface ocean.

Genomic and functional adaptation in surface ocean planktonic prokaryotes.

The understanding of marine microbial ecology and metabolism has been hampered by the paucity of sequenced reference genomes. To this end, we report the sequencing of 137 diverse marine isolates collected from around the world. We analysed these sequences, along with previously published marine prokaryotic genomes, in the context of marine metagenomic data, to gain insights into the ecology of the surface ocean prokaryotic picoplankton (0.

Chemical synthesis of the mouse mitochondrial genome.

We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.

Characterization of Prochlorococcus clades from iron-depleted oceanic regions.

Prochlorococcus describes a diverse and abundant genus of marine photosynthetic microbes. It is primarily found in oligotrophic waters across the globe and plays a crucial role in energy and nutrient cycling in the ocean ecosystem. The abundance, global distribution, and availability of isolates make Prochlorococcus a model system for understanding marine microbial diversity and biogeochemical cycling.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle.

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb.

Creation of a bacterial cell controlled by a chemically synthesized genome.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M.

Towards a comprehensive structural variation map of an individual human genome.

Background: Several genomes have now been sequenced, with millions of genetic variants annotated. While significant progress has been made in mapping single nucleotide polymorphisms (SNPs) and small (<10 bp) insertion/deletions (indels), the annotation of larger structural variants has been less comprehensive. It is still unclear to what extent a typical genome differs from the reference assembly, and the analysis of the genomes sequenced to date have shown varying results for copy number variation (CNV) and inversions.

The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration.

Cloning whole bacterial genomes in yeast.

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules.

Creating bacterial strains from genomes that have been cloned and engineered in yeast.

We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this.

Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina.

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T.

Enzymatic assembly of DNA molecules up to several hundred kilobases.

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line.

We have identified new genomic alterations in the breast cancer cell line HCC1954, using high-throughput transcriptome sequencing. With 120 Mb of cDNA sequences, we were able to identify genomic rearrangement events leading to fusions or truncations of genes including MRE11 and NSD1, genes already implicated in oncogenesis, and 7 rearrangements involving other additional genes. This approach demonstrates that high-throughput transcriptome sequencing is an effective strategy for the characterization of genomic rearrangements in cancers.

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome.

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step.