Roger S. Lasken

Comprehensive human genome amplification using multiple displacement amplification

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

Mosaic Copy Number Variation in Human Neurons

Not All Neurons Are Alike As life proceeds, many cells acquire individualized mutations. In the immune system, genome rearrangements generate useful antibody diversity. McConnell et al. (p. 632; see the Perspective by Macosko and McCarroll) now show that human neurons also diversify. Neurons taken from postmortem human frontal cortex tissue and neurons derived from induced pluripotent stem cell differentiation in vitro showed surprising diversity in individual cell genomes. Up to 41% of the frontal cortex neurons had copy number variations—no two alike—with deletions more common than duplications. Single-cell genomics reveals that individual adult human neurons acquire diverse individual genomes. [Also see Perspective by Macosko and McCarroll] We used single-cell genomic approaches to map DNA copy number variation (CNV) in neurons obtained from human induced pluripotent stem cell (hiPSC) lines and postmortem human brains. We identified aneuploid neurons, as well as numerous subchromosomal CNVs in euploid neurons. Neurotypic hiPSC-derived neurons had larger CNVs than fibroblasts, and several large deletions were found in hiPSC-derived neurons but not in matched neural progenitor cells. Single-cell sequencing of endogenous human frontal cortex neurons revealed that 13 to 41% of neurons have at least one megabase-scale de novo CNV, that deletions are twice as common as duplications, and that a subset of neurons have highly aberrant genomes marked by multiple alterations. Our results show that mosaic CNV is abundant in human neurons.

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. Phylogenomic analyses establish SAR86 as a basal and divergent lineage of γ-proteobacteria, and the individual genomes display a temperature-dependent distribution. Modestly sized at 1.25–1.7 Mbp, the SAR86 genomes lack several pathways for amino-acid and vitamin synthesis as well as sulfate reduction, trends commonly observed in other abundant marine microbes. SAR86 appears to be an aerobic chemoheterotroph with the potential for proteorhodopsin-based ATP generation, though the apparent lack of a retinal biosynthesis pathway may require it to scavenge exogenously-derived pigments to utilize proteorhodopsin. The genomes contain an expanded capacity for the degradation of lipids and carbohydrates acquired using a wealth of tonB-dependent outer membrane receptors. Like the abundant planktonic marine bacterial clade SAR11, SAR86 exhibits metabolic streamlining, but also a distinct carbon compound specialization, possibly avoiding competition.

Genomic DNA Amplification from a Single Bacterium

ABSTRACT Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using φ 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.

Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Targeted metagenomics and ecology of globally important uncultured eukaryotic phytoplankton

Among eukaryotes, four major phytoplankton lineages are responsible for marine photosynthesis; prymnesiophytes, alveolates, stramenopiles, and prasinophytes. Contributions by individual taxa, however, are not well known, and genomes have been analyzed from only the latter two lineages. Tiny “picoplanktonic” members of the prymnesiophyte lineage have long been inferred to be ecologically important but remain poorly characterized. Here, we examine pico-prymnesiophyte evolutionary history and ecology using cultivation-independent methods. 18S rRNA gene analysis showed pico-prymnesiophytes belonged to broadly distributed uncultivated taxa. Therefore, we used targeted metagenomics to analyze uncultured pico-prymnesiophytes sorted by flow cytometry from subtropical North Atlantic waters. The data reveal a composite nuclear-encoded gene repertoire with strong green-lineage affiliations, which contrasts with the evolutionary history indicated by the plastid genome. Measured pico-prymnesiophyte growth rates were rapid in this region, resulting in primary production contributions similar to the cyanobacterium Prochlorococcus. On average, pico-prymnesiophytes formed 25% of global picophytoplankton biomass, with differing contributions in five biogeographical provinces spanning tropical to subpolar systems. Elements likely contributing to success include high gene density and genes potentially involved in defense and nutrient uptake. Our findings have implications reaching beyond pico-prymnesiophytes, to the prasinophytes and stramenopiles. For example, prevalence of putative Ni-containing superoxide dismutases (SODs), instead of Fe-containing SODs, seems to be a common adaptation among eukaryotic phytoplankton for reducing Fe quotas in low-Fe modern oceans. Moreover, highly mosaic gene repertoires, although compositionally distinct for each major eukaryotic lineage, now seem to be an underlying facet of successful marine phytoplankton.

Efficient de novo assembly of single-cell bacterial genomes from short-read data sets

Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of DNA from single cells of bacteria that cannot be cultured. Assembling a genome is challenging, however, because MDA generates highly nonuniform coverage of the genome. Here we describe an algorithm tailored for short-read data from single cells that improves assembly through the use of a progressively increasing coverage cutoff. Assembly of reads from single Escherichia coli and Staphylococcus aureus cells captures >91% of genes within contigs, approaching the 95% captured from an assembly based on many E. coli cells. We apply this method to assemble a genome from a single cell of an uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. Our approach enables acquisition of genome assemblies for individual uncultivated bacteria using only short reads, providing cell-specific genetic information absent from metagenomic studies.Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of genomes from single cells of bacteria that cannot be cultured. However, genome assembly is challenging because of highly non-uniform read coverage generated by MDA. We describe an improved assembly approach tailored for single cell Illumina sequences that incorporates a progressively increasing coverage cutoff. This allows variable coverage datasets to be utilized effectively with assembly of E. coli and S. aureus single cell reads capturing >91% of genes within contigs, approaching the 95% captured from a multi-cell E. coli assembly. We apply this method to assemble a single cell genome of the uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. These new methods enable acquisition of genome assemblies for individual uncultivated bacteria, providing cell-specific genetic information absent from metagenomic studies.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a ‘power tool’ in the molecular toolbox of microbial ecologists.

Insights into the genome of large sulfur bacteria revealed by analysis of single filaments.

Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical importance, little is known about their genetic repertoire because of the lack of pure cultures. Here, we present a unique approach to access the genome of single filaments of Beggiatoa by combining whole genome amplification, pyrosequencing, and optical genome mapping. Sequence assemblies were incomplete and yielded average contig sizes of approximately 1 kb. Pathways for sulfur oxidation, nitrate and oxygen respiration, and CO2 fixation confirm the chemolithoautotrophic physiology of Beggiatoa. In addition, Beggiatoa potentially utilize inorganic sulfur compounds and dimethyl sulfoxide as electron acceptors. We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur and nitrogen cycling in marine sediments.

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

Significance This research highlights the discovery and genome reconstruction of a member of the globally distributed yet uncultivated candidate phylum TM6 (designated TM6SC1). In addition to the 16S rRNA gene, no other genomic information is available for this cosmopolitan phylum. This report also introduces a mini-metagenomic approach based on the use of high-throughput single-cell genomics techniques and assembly tools that address a widely recognized issue: how to effectively capture and sequence the currently uncultivated bacterial species that make up the “dark matter of life.” Amplification and sequencing random pools of 100 events enabled an estimated 90% recovery of the TM6SC1 genome. 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. We developed an approach for increasing throughput and effectively improving the likelihood of sampling rare events based on forming small random pools of single-flow–sorted cells, amplifying their DNA by multiple displacement amplification and sequencing all cells in the pool, creating a “mini-metagenome.” A recently developed single-cell assembler, SPAdes, in combination with contig binning methods, allowed the reconstruction of genomes from these mini-metagenomes. A total of 1.07 Mb was recovered in seven contigs for this member of TM6 (JCVI TM6SC1), estimated to represent 90% of its genome. High nucleotide identity between a total of three TM6 genome drafts generated from pools that were independently captured, amplified, and assembled provided strong confirmation of a correct genomic sequence. TM6 is likely a Gram-negative organism and possibly a symbiont of an unknown host (nonfree living) in part based on its small genome, low-GC content, and lack of biosynthesis pathways for most amino acids and vitamins. Phylogenomic analysis of conserved single-copy genes confirms that TM6SC1 is a deeply branching phylum.