Selective Whole Genome Amplification for Resequencing Target Microbial Species from Complex Natural Samples

Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host–microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10⁵-fold amplification of the target genomes with <6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2–9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs.     

Using partial genomic fosmid libraries for sequencing complete organellar genomes

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.     

CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes

The genomes are organized into ordered and hierarchical topological structures in interphase nuclei.Within discrete territories of each chromosome,topologically associated domains(TADs) play important roles in various nuclear processes such as gene regulation.Inside TADs separated by relatively constitutive boundaries,distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions.High-throughput sequencing studies have revealed millions of potential regulatory DNA elements,which are much more abundant than the mere ~ 20,000 genes they control.The recently emerged CRISPRCas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes.The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements.Here,we describe the applications of CRISPR for genome,epigenome,and 3D genome editing,focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.     

TERAD: Extraction of transposable element composition from RADseq data

Transposable elements (TEs) – selfish DNA sequences that can move within the genome – comprise a large proportion of the genomes of many organisms. Although low‐coverage whole‐genome sequencing can be used to survey TE composition, it is noneconomical for species with large quantities of DNA. Here, we utilize restriction‐site associated DNA sequencing (RADSeq) as an alternative method to survey TE composition. First, we demonstrate in silico that double digest restriction‐site associated DNA sequencing (ddRADseq) markers contain the same TE compositions as whole genome assemblies across arthropods. Next, we show empirically using eight Synalpheus snapping shrimp species with large genomes that TE compositions from ddRADseq and low‐coverage whole‐genome sequencing are comparable within and across species. Finally, we develop a new bioinformatic pipeline, TERAD, to extract TE compositions from RADseq data. Our study expands the utility of RADseq to study the repeatome, making comparative studies of genome structure for species with large genomes more tractable and affordable.     

Model genomes: the benefits of analysing homologous human and mouse sequences.

The human genome initiative has provided the motivating force for launching sequencing projects suitable for testing various DNA-sequencing strategies, as well as motivating the development of mapping and sequencing technologies. In addition to projects targeting selected regions of the human genome, other projects are based on model organisms such as yeast, nematode and mouse. The sequencing of homologous regions of human and mouse genomes is a new approach to genome analysis, and is providing insights into gene evolution, function and regulation which could not be determined so easily from the analysis of just one species.     

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses

Background

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change.

Methodology/Principal Findings

We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses.

Conclusions/Significance

Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.     

双翅目昆虫线粒体基因组结构特点及其测序通用引物的设计和应用

研究双翅目昆虫线粒体基因组的结构特点, 并设计其测序的通用引物, 为今后双翅目昆虫线粒体基因组的研究提供参考和依据。利用比较基因组学和生物信息学方法, 分析了已经完全测序的26个双翅目昆虫线粒体基因组的结构特点、 碱基组成和保守区, 并据此设计了双翅目昆虫基因组测序的通用引物。结果表明： 双翅目昆虫线粒体基因组长14 503~19 517 bp, 其结构保守, 含有37个编码基因, 包括13个蛋白质编码基因, 22个tRNA编码基因和2个rRNA编码基因, 此外还包含一段长度差异很大的非编码区(AT富含区)。基因组内基因排列次序稳定, 除个别基因外, 其余都与黑腹果蝇Drosophila melanogaster基因排列次序一致。基因组的碱基组成不均衡, AT含量在72.59%~85.15%之间, 碱基使用存在偏向性, 偏好使用AC碱基。全基因组的核苷酸和氨基酸序列保守, 共鉴定了11个保守区。在保守区内共设计了26对双翅目线粒体基因组测序通用引物, 扩增的目标片段都在1 200 bp以内。将该套通用引物用于葱蝇Delia antiqua线粒体全基因组测序, 结果证明其高效、 合用。     

Methods for identification of epigenetic elements in mammalian long multigenic genome sequences

Epigenetic elements of the genome, i.e. elements that determine stably inherited changes in gene expression without changes in the genomic DNA sequence, are essential tools of genetic regulation in higher eukaryotes. The complete sequencing of the human and other genomes allowed studies to be started on positioning of these elements within long multigenic regions of the genome, which is a prerequisite for a comprehensive functional annotation of genomes. This mini-review considers some recent experimental approaches to the high-throughput identification and mapping of epigenetic elements of mammalian genomes, including the mapping of methylated CpG sites, open and closed chromatin regions, and DNase I hypersensitivity sites.     

What transposable elements tell us about genome organization and evolution: the case of Drosophila

Transposable elements (TEs) have been identified in every organism in which they have been looked for. The sequencing of large genomes, such as the human genome and those of Drosophila, Arabidopsis, Caenorhabditis, has also shown that they are a major constituent of these genomes, accounting for 15% of the genome of Drosophila, 45% of the human genome, and more than 70% in some plants and amphibians. Compared with the 1% of genomic DNA dedicated to protein-coding sequences in the human genome, this has prompted various researchers to suggest that the TEs and the other repetitive sequences that constitute the so-called "noncoding DNA", are where the most stimulating discoveries will be made in the future (Bromham, 2002). We are therefore getting further and further from the original idea that this DNA was simply "junk DNA", that owed its presence in the genome entirely to its capacity for selfish transposition. Our understanding of the structures of TEs, their distribution along the genomes, their sequence and insertion polymorphisms within genomes, and within and between populations and species, their impact on genes and on the regulatory mechanisms of genetic expression, their effects on exon shuffling and other phenomena that reshape the genome, and their impact on genome size has increased dramatically in recent years. This leads to a more general picture of the impact of TEs on genomes, though many copies are still mainly selfish or junk DNA. In this review we focus mainly on discoveries made in Drosophila, but we also use information about other genomes when this helps to elucidate the general processes involved in the organization, plasticity, and evolution of genomes.     

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.     

真菌基因组de novo测序组装的方法与实践

真菌基因组较其他真核生物基因组结构简单,长度短,易于测序、组装与注释,因此真菌基因组是研究真核生物基因组的模型。为研究真菌基因组组装策略,本研究基于Illumina HiSeq测序平台对烟曲霉菌株An16007基因组测序,分别使用5种de novo组装软件ABySS、SOAP-denovo、Velvet、MaSuRCA和IDBA-UD组装基因组,然后通过Augustus软件进行基因预测,BUSCO软件评估组装结果。研究发现,5种组装软件对基因组组装结果不同,ABySS组装的基因组较其他4种组装软件具有更高的完整性和准确性,且预测的基因数量较高,因此,ABySS更适合本研究基因组的组装。本研究提供了真菌de novo测序、组装及组装质量评估的技术流程,为基因组<100 Mb的真菌或其他生物基因组的研究提供参考。     

Reduced representation sequencing: a success in maize and a promise for other plant genomes

Plant, and particularly cereal genomes, are challenging to sequence due to their large size and high repetitive DNA content. Gene-enrichment strategies are alternative or complementary approaches to complete genome sequencing that yield, rapidly and inexpensively, useful sequence data from large and complex genomes. The maize genome is large (2.7 Gbp) and contains large amounts of conserved repetitive elements. Furthermore, the high allelic diversity found between maize inbred lines may necessitate sequencing several inbred lines in order to recover the maize "gene pool". Two gene-enrichment approaches, methylation filtration (MF) and high C(o)t (HC) sequencing have been tested in maize and their ability to sample the gene space has been examined. Combined with other genomic sequencing strategies, gene-enriched genomic sequencing is a practical way to examine the maize gene pool, to order and orient the genic sequences on the genome, and to enable investigation of gene content of other complex plant genomes.     

Characterization of the ELPhiS prophage from Salmonella enterica serovar Enteritidis strain LK5

Phages are a primary driving force behind the evolution of bacterial pathogens by transferring a variety of virulence genes into their hosts. Similar to other bacterial genomes, the Salmonella enterica serovar Enteritidis LK5 genome contains several regions that are homologous to phages. Although genomic analysis demonstrated the presence of prophages, it was unable to confirm which phage elements within the genome were viable. Genetic markers were used to tag one of the prophages in the genome to allow monitoring of phage induction. Commonly used laboratory strains of Salmonella were resistant to phage infection, and therefore a rapid screen was developed to identify susceptible hosts. This approach showed that a genetically tagged prophage, ELPhiS (Enteritidis lysogenic phage S), was capable of infecting Salmonella serovars that are diverse in host range and virulence and has the potential to laterally transfer genes between these serovars via lysogenic conversion. The rapid screen approach is adaptable to any system with a large collection of isolates and may be used to test the viability of prophages found by sequencing the genomes of various bacterial pathogens.     

A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies

With the expansion of next‐generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost‐effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next‐generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large‐scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies.     

Mutation detection with next-generation resequencing through a mediator genome

The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.     

Give-and-take: interactions between DNA transposons and their host plant genomes

Recent genome sequencing efforts have revealed how extensively transposable elements (TEs) have contributed to the shaping of present day plant genomes. DNA transposons associate preferentially with the euchromatic or genic component of plant genomes and have had the opportunity to interact intimately with the genes of the plant host. These interactions have resulted in TEs acquiring host sequences, forming chimeric genes through exon shuffling, replacing regulatory sequences, mobilizing genes around the genome, and contributing genes to the host. The close interaction of transposons with genes has also led to the evolution of intricate cellular mechanisms for silencing transposon activity. Transposons have thus become important subjects of study in understanding epigenetic regulation and, in cases where transposons have amplified to high numbers, how to escape that regulation.