Benchmarking of single-virus genomics: a new tool for uncovering the virosphere

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.     

微生物单细胞基因组技术及其在环境微生物研究中的应用

单细胞及单细胞基因组学研究是近年生命科学研究的热点之一,微生物单细胞基因组学研究是继微生物元基因组学(又称宏基因组学,Metagenomics)之后新发展起来的,可有效获取环境中大量无法培养的微生物遗传信息的技术。微生物单细胞基因组技术包括单细胞获取、全基因组扩增、全基因组测序以及数据分析等步骤,目前该技术在环境微生物研究中的应用主要集中于探索未被元基因组技术或其它常规技术探测到的新型功能基因,或是对环境中物种丰度极小的未培养微生物的发现,以及对微生物细胞生命进化过程的研究等。本文对微生物单细胞基因组技术中单细胞获取和全基因组扩增所涉及到的不同方法以及应用此技术对环境微生物取得的主要研究进展进行综述。     

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation ^1-3. Viruses, which are ubiquitous and the most numerous entities on our planet ⁴ and important in all environments ⁵, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called ''Single Virus Genomics'' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.     

Improved Bacteriophage Genome Data is Necessary for Integrating Viral and Bacterial Ecology

The recent rise in “omics”-enabled approaches has lead to improved understanding in many areas of microbial ecology. However, despite the importance that viruses play in a broad microbial ecology context, viral ecology remains largely not integrated into high-throughput microbial ecology studies. A fundamental hindrance to the integration of viral ecology into omics-enabled microbial ecology studies is the lack of suitable reference bacteriophage genomes in reference databases—currently, only 0.001 % of bacteriophage diversity is represented in genome sequence databases. This commentary serves to highlight this issue and to promote bacteriophage genome sequencing as a valuable scientific undertaking to both better understand bacteriophage diversity and move towards a more holistic view of microbial ecology.     

New DNA viruses identified in patients with acute viral infection syndrome

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.     

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.     

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.     

One Bacterial Cell,One Complete Genome

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200–900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.     

From deep sequencing to viral tagging: Recent advances in viral metagenomics

Culture‐independent high‐throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non‐viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high‐throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation‐independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts.     

Functional genomics as a tool in virus research

Genomics is the study of an organism’s entire genome. It started out as a great scientific endeavor in the 1990s which aimed to sequence the complete genomes of certain biological species. However viruses are not new to this field as complete viral genomes have routinely been sequenced since the past thirty years. The ‘genomic era’ has been said to have revolutionized biology. This knowledge of full genomes has created the field of functional genomics in today’s post-genomic era, which, is in most part concerned with the studies on the expression of the organism’s genome under different conditions. This article is an attempt to introduce its readers to the application of functional genomics to address and answer several complex biological issues in virus research.     

Aquatic virus diversity accessed through omic techniques: a route map to function

Viruses are arguably the simplest form of life yet they play a crucial role in regulating planetary processes. From shuttling genes to 'lubricating' microbial loop dynamics, viruses are integral in shaping microbial ecology. In every environment on Earth the role of viruses goes far beyond the simple infect-replicate-kill cycle. Their enormous abundance and seemingly infinite diversity provide the vital clues to the true function of viruses. New 'omic' approaches are now allowing researchers to gain extraordinary insights into virus diversity and inferred function, particularly within aquatic environments. The development of molecular markers and application of techniques including microarrays, metagenomic sequencing and proteomic analysis are now being applied to virus communities. Despite this shift towards culture-independent approaches it has proved difficult to derive useful information about infection strategies since so much of the sequence information has no database matches. Future advances will involve tools such as microarrays to help determine the functionality of unknown genes. Sequence information should be considered as a starting point for asking questions and developing hypotheses about the role of viruses. It is an exciting new era for virus ecology and when used in combination with more traditional approaches, virus genomics will give us access to their ecological function on an unprecedented scale.     

From bacterial genomics to metagenomics: concept,tools and recent advances

In the last 20 years, the applications of genomics tools have completely transformed the field of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, first describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting findings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world. The authors have contributed equally to the work     

Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances

Microbial ecology examines the diversity and activity of micro-organisms in Earth's biosphere. In the last 20 years, the application of genomics tools have revolutionized microbial ecological studies and drastically expanded our view on the previously underappreciated microbial world. This review first introduces the basic concepts in microbial ecology and the main genomics methods that have been used to examine natural microbial populations and communities. In the ensuing three specific sections, the applications of the genomics in microbial ecological research are highlighted. The first describes the widespread application of multilocus sequence typing and representational difference analysis in studying genetic variation within microbial species. Such investigations have identified that migration, horizontal gene transfer and recombination are common in natural microbial populations and that microbial strains can be highly variable in genome size and gene content. The second section highlights and summarizes the use of four specific genomics methods (phylogenetic analysis of ribosomal RNA, DNA-DNA re-association kinetics, metagenomics, and micro-arrays) in analysing the diversity and potential activity of microbial populations and communities from a variety of terrestrial and aquatic environments. Such analyses have identified many unexpected phylogenetic lineages in viruses, bacteria, archaea, and microbial eukaryotes. Functional analyses of environmental DNA also revealed highly prevalent, but previously unknown, metabolic processes in natural microbial communities. In the third section, the ecological implications of sequenced microbial genomes are briefly discussed. Comparative analyses of prokaryotic genomic sequences suggest the importance of ecology in determining microbial genome size and gene content. The significant variability in genome size and gene content among strains and species of prokaryotes indicate the highly fluid nature of prokaryotic genomes, a result consistent with those from multilocus sequence typing and representational difference analyses. The integration of various levels of ecological analyses coupled to the application and further development of high throughput technologies are accelerating the pace of discovery in microbial ecology.     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

单细胞全基因组扩增技术与应用

同一组织中的细胞往往具有类似的结构和功能,然而通过对单个细胞进行测序分析后,发现每个细胞都具有一定异质性.单细胞全基因组扩增技术是进行单细胞测序的前提,该技术可用于揭示单细胞基因组结构差异,同时在肿瘤研究、发育生物学、微生物学等研究中发挥重要作用,并成为生命科学研究技术的热点之一.单细胞全基因组扩增技术的难点在于单细胞的分离和全基因组的扩增.本文介绍了单细胞全基因组扩增技术中常用的单细胞分离技术和单细胞全基因组扩增技术,并对各技术间的优缺点进行比较,同时着重讨论该技术在肿瘤研究、发育生物学和微生物学研究中的应用.     

Widespread Recombination,Reassortment, and Transmission of Unbalanced Compound Viral Genotypes in Natural Arenavirus Infections

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.