Shotgun sequencing,clone pooling,and comparative strategies for mapping and sequencing

Comparative genomic sequencing and analysis offers new wealth of information for target selection and the development of therapeutics. This article focuses on the following two key innovations in mapping and sequencing: first, shotgun sequencing of clone pools to combine the benefits of whole-genome shotgun and clone-by-clone strategies, and second, the leveraging of newly available assembled genomic sequences to improve the effectiveness of new sequencing projects through comparative mapping and comparative sequence assembly. The following specific sequencing and mapping methods are discussed in detail: clone-array pooled shotgun sequencing (CAPSS); transversal shotgun pooling designs; clone-array pooled shotgun mapping (CAPS-MAP); pooled genomic indexing (PGI); short-tag pooled genomic indexing (ST-PGI); and comparative sequence assembly (the CSA™ method). The methods can be implemented with only modest modifications of current large-scale sequencing pipelines and are highly synergistic with the next generation of sequencing technologies.     

四种常用高通量测序拼接软件的应用比较

新一代测序平台的诞生推动了对全基因组鸟枪法测序数据的拼接算法和软件的研究,自2005年以来多种用于高通量测序的序列拼接软件已经被开发出来,并且在不断地进行改进以提高拼接效果.本文利用目前广泛使用的高通量测序拼接软件Velvet、AbySS、SOAPdenovo和CLC Genomic Workbench分别对本试验室分离的一株噬菌体IME08的高通量测序结果进行拼接,介绍这几种拼接软件的安装使用及参数优化,并对不同软件的拼接结果进行比较,针对不同的拼接软件得到优化的拼接参数,可为其他研究人员使用上述软件提供参考借鉴.     

Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation

The fungal diversity in deep-sea environments has recently gained an increasing amount attention. Our knowledge and understanding of the true fungal diversity and the role it plays in deep-sea environments, however, is still limited. We investigated the fungal community structure in five sediments from a depth of ∼4000 m in the East India Ocean using a combination of targeted environmental sequencing and traditional cultivation. This approach resulted in the recovery of a total of 45 fungal operational taxonomic units (OTUs) and 20 culturable fungal phylotypes. This finding indicates that there is a great amount of fungal diversity in the deep-sea sediments collected in the East Indian Ocean. Three fungal OTUs and one culturable phylotype demonstrated high divergence (89%–97%) from the existing sequences in the GenBank. Moreover, 44.4% fungal OTUs and 30% culturable fungal phylotypes are new reports for deep-sea sediments. These results suggest that the deep-sea sediments from the East India Ocean can serve as habitats for new fungal communities compared with other deep-sea environments. In addition, different fungal community could be detected when using targeted environmental sequencing compared with traditional cultivation in this study, which suggests that a combination of targeted environmental sequencing and traditional cultivation will generate a more diverse fungal community in deep-sea environments than using either targeted environmental sequencing or traditional cultivation alone. This study is the first to report new insights into the fungal communities in deep-sea sediments from the East Indian Ocean, which increases our knowledge and understanding of the fungal diversity in deep-sea environments.     

Archaeal diversity and community development in deep-sea hydrothermal vents

Over the past 35 years, researchers have explored deep-sea hydrothermal vent environments around the globe and studied a number of archaea, their unique metabolic and physiological properties, and their vast phylogenetic diversity. Although the pace of discovery of new archaeal taxa, phylotypes and phenotypes in deep-sea hydrothermal vents has slowed recently, bioinformatics and interdisciplinary geochemistry-microbiology approaches are providing new information on the diversity and community composition of archaea living in deep-sea vents. Recent investigations have revealed that archaea could have originated and dispersed from ancestral communities endemic to hydrothermal vents into other biomes on Earth, and the community structure and productivity of chemolithotrophic archaea are controlled primarily by variations in the geochemical composition of hydrothermal fluids.     

Annotated Genome Sequence of Mycobacterium massiliense Strain M154, Belonging to the Recently Created Taxon Mycobacterium abscessus subsp. bolletii comb. nov

Mycobacterium massiliense has recently been proposed as a member of Mycobacterium abscessus subsp. bolletii comb. nov. Strain M154, a clinical isolate from the bronchoalveolar lavage fluid of a Malaysian patient presenting with lower respiratory tract infection, was subjected to shotgun DNA sequencing with the Illumina sequencing technology to obtain whole-genome sequence data for comparison with other genetically related strains within the M. abscessus species complex.     

Exploring the composition and diversity of microbial communities at the Jan Mayen hydrothermal vent field using RNA and DNA

DNA sequencing technology has proven very valuable for analysing the microbiota of poorly accessible ecosystems such as hydrothermal vents. Using a combination of amplicon and shotgun sequencing of small-subunit rRNA and its gene, we examined the composition and diversity of microbial communities from the recently discovered Jan Mayen vent field, located on Mohn's Ridge in the Norwegian-Greenland Sea. The communities were dominated by the epsilonproteobacterial genera Sulfurimonas and Sulfurovum. These are mesophiles involved in sulphur metabolism and typically found in vent fluid mixing zones. Composition and diversity predictions differed systematically between extracted DNA and RNA samples as well as between amplicon and shotgun sequencing. These differences were more substantial than those between two biological replicates. Amplicon vs. shotgun sequencing differences could be explained to a large extent by bias introduced during PCR, caused by preferential primer-template annealing, while DNA vs. RNA differences were thought to be caused by differences between the activity levels of taxa. Further, predicted diversity from RNA samples was consistently lower than that from DNA. In summary, this study illustrates how different methods can provide complementary ecological insights.     

Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data

The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%–5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.     

Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.     

Is metagenomics resolving identification of functions in microbial communities?

We are coming up on the tenth anniversary of the broad use of the method involving whole metagenome shotgun sequencing, referred to as metagenomics. The application of this approach has definitely revolutionized microbiology and the related fields, including the realization of the importance of the human microbiome. As such, metagenomics has already provided a novel outlook on the complexity and dynamics of microbial communities that are an important part of the biosphere of the planet. Accumulation of massive amounts of sequence data also caused a surge in the development of bioinformatics tools specially designed to provide pipelines for data analysis and visualization. However, a critical outlook into the field is required to appreciate what could be and what has currently been gained from the massive sequence databases that are being generated with ever‐increasing speed.     

Sampling,sequencing and the SAD

The Species Abundance Distribution (SAD) is a common metric for characterizing macroscopic ecological communities. Recently, this metric has been applied to analysis of microbial communities as well. However, as compared to macroscopic communities, sampling of microscopic communities is different. In particular, most microbial communities are studied using sequencing techniques. These techniques have known biases that result in certain taxa being detected more often than others, even if the taxa are present in the sample at equivalent abundances. There are, for example, amplification biases that result in some sequences being amplified more than others. Likewise, differences in genome size across organisms can result in different numbers of reads from different taxa, again resulting in biased detection. A number of bioinformatics methods have been devised to account for biases in sequencing data, allowing for more accurate estimates of relative taxon abundances. However, because the sampling process itself is affected by biased detection, and because sampling (and under-sampling in particular) can influence the shape of the SAD, it is possible that, even when corrected for through re-scaling, detection biases can affect SAD predictions from sequencing data. To test this hypothesis, we construct a simulation model of the sampling process, focusing on biased detection in shotgun sequencing that arises from genome size differences across microbial taxa. Interestingly, we find that, although genome size itself does not impact SAD predictions, predictions can vary depending on the range of genome sizes that are represented in a community, as well as how genome size is distributed (i.e., whether the majority of species have small versus large genomes). Our results suggest that care should be taken when comparing SADs across environments, particularly when those environments might have taxa with different genome size distributions. Furthermore, our results indicate that relatively deep sequencing might be required to avoid drawing spurious inferences about ecological differences across microbial communities.     

De novo discovery and multiplexed amplification of microsatellite markers for black alder (Alnus glutinosa) and related species using SSR-enriched shotgun pyrosequencing

Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in nonmodel organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17?855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, 12 of which were polymorphic in A. glutinosa. These 12 loci were successfully coamplified in a single multiplex polymerase chain reaction experiment and validated for population genetics applications. In addition, 10 and 8 of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.     

Harvesting the mouse genome

The sequencing of the black 6 mouse (strain C57Bl/6) has reached an important juncture. The BAC fingerprint map is almost complete, the BACs have been endsequenced and a seven-fold coverage whole-genome shotgun has been assembled. Now the BAC-by-BAC sequencing phase is under way and in-depth comparative analysis can be carried out on regions that have been the subject of targeted sequencing. This paper reviews the progress so far and looks forward to the promises of finished sequence.     

Resources and costs for microbial sequence analysis evaluated using virtual machines and cloud computing

Background

The widespread popularity of genomic applications is threatened by the “bioinformatics bottleneck” resulting from uncertainty about the cost and infrastructure needed to meet increasing demands for next-generation sequence analysis. Cloud computing services have been discussed as potential new bioinformatics support systems but have not been evaluated thoroughly.

Results

We present benchmark costs and runtimes for common microbial genomics applications, including 16S rRNA analysis, microbial whole-genome shotgun (WGS) sequence assembly and annotation, WGS metagenomics and large-scale BLAST. Sequence dataset types and sizes were selected to correspond to outputs typically generated by small- to midsize facilities equipped with 454 and Illumina platforms, except for WGS metagenomics where sampling of Illumina data was used. Automated analysis pipelines, as implemented in the CloVR virtual machine, were used in order to guarantee transparency, reproducibility and portability across different operating systems, including the commercial Amazon Elastic Compute Cloud (EC2), which was used to attach real dollar costs to each analysis type. We found considerable differences in computational requirements, runtimes and costs associated with different microbial genomics applications. While all 16S analyses completed on a single-CPU desktop in under three hours, microbial genome and metagenome analyses utilized multi-CPU support of up to 120 CPUs on Amazon EC2, where each analysis completed in under 24 hours for less than $60. Representative datasets were used to estimate maximum data throughput on different cluster sizes and to compare costs between EC2 and comparable local grid servers.

Conclusions

Although bioinformatics requirements for microbial genomics depend on dataset characteristics and the analysis protocols applied, our results suggests that smaller sequencing facilities (up to three Roche/454 or one Illumina GAIIx sequencer) invested in 16S rRNA amplicon sequencing, microbial single-genome and metagenomics WGS projects can achieve cost-efficient bioinformatics support using CloVR in combination with Amazon EC2 as an alternative to local computing centers.     

RiboTagger: fast and unbiased 16S/18S profiling using whole community shotgun metagenomic or metatranscriptome surveys

Background

Taxonomic profiling of microbial communities is often performed using small subunit ribosomal RNA (SSU) amplicon sequencing (16S or 18S), while environmental shotgun sequencing is often focused on functional analysis. Large shotgun datasets contain a significant number of SSU sequences and these can be exploited to perform an unbiased SSU--based taxonomic analysis.

Results

Here we present a new program called RiboTagger that identifies and extracts taxonomically informative ribotags located in a specified variable region of the SSU gene in a high-throughput fashion.

Conclusions

RiboTagger permits fast recovery of SSU-RNA sequences from shotgun nucleic acid surveys of complex microbial communities. The program targets all three domains of life, exhibits high sensitivity and specificity and is substantially faster than comparable programs.

     

Microbial community analysis using high-throughput sequencing technology: a beginner’s guide for microbiologists

Microbial communities present in diverse environments from deep seas to human body niches play significant roles in the complex ecosystem and human health. Characterizing their structural and functional diversities is indispensable, and many approaches, such as microscopic observation, DNA fingerprinting, and PCR-based marker gene analysis, have been successfully applied to identify microorganisms. Since the revolutionary improvement of DNA sequencing technologies, direct and high-throughput analysis of genomic DNA from a whole environmental community without prior cultivation has become the mainstream approach, overcoming the constraints of the classical approaches. Here, we first briefly review the history of environmental DNA analysis applications with a focus on profiling the taxonomic composition and functional potentials of microbial communities. To this end, we aim to introduce the shotgun metagenomic sequencing (SMS) approach, which is used for the untargeted (“shotgun”) sequencing of all (“meta”) microbial genomes (“genomic”) present in a sample. SMS data analyses are performed in silico using various software programs; however, in silico analysis is typically regarded as a burden on wet-lab experimental microbiologists. Therefore, in this review, we present microbiologists who are unfamiliar with in silico analyses with a basic and practical SMS data analysis protocol. This protocol covers all the bioinformatics processes of the SMS analysis in terms of data preprocessing, taxonomic profiling, functional annotation, and visualization.     

基于宏组学方法认识微生物群落及其功能

进入后基因组学时代,测序技术飞速发展,测序成本明显下降,形成了涵盖宏基因组学、宏转录组学和宏蛋白质组学的宏组学技术,推动了对微生物群落的多样性、结构及潜在基因功能方面的深入研究。最近随着整合的宏组学技术的提出及应用,全面系统分析微生物群落动态变化及其代谢功能已成为可能,这将成为微生物生态学研究的新趋势。本文综述了宏组学在研究海洋湖泊、深海热泉、人体肠道、牛瘤胃生境、森林土壤与堆肥生境等环境中微生物群落的结构和功能方面的最新进展与成功应用案例。     

Choosing the Best Plant for the Job: A Cost-Effective Assay to Prescreen Ancient Plant Remains Destined for Shotgun Sequencing

DNA extracted from ancient plant remains almost always contains a mixture of endogenous (that is, derived from the plant) and exogenous (derived from other sources) DNA. The exogenous ‘contaminant’ DNA, chiefly derived from microorganisms, presents significant problems for shotgun sequencing. In some samples, more than 90% of the recovered sequences are exogenous, providing limited data relevant to the sample. However, other samples have far less contamination and subsequently yield much more useful data via shotgun sequencing. Given the investment required for high-throughput sequencing, whenever multiple samples are available, it is most economical to sequence the least contaminated sample. We present an assay based on quantitative real-time PCR which estimates the relative amounts of fungal and bacterial DNA in a sample in comparison to the endogenous plant DNA. Given a collection of contextually-similar ancient plant samples, this low cost assay aids in selecting the best sample for shotgun sequencing.