The genome of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius does not contain repetitive sequences

Characteristics of genome organization in the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied. By means of hybridization analysis it is shown that the genome of S. acidocaldarius, unlike the genome of the extremely halophilic archaebacterium Halobacterium halobium, does not contain repetitive sequences.     

CAR: contig assembly of prokaryotic draft genomes using rearrangements

Background

Next generation sequencing technology has allowed efficient production of draft genomes for many organisms of interest. However, most draft genomes are just collections of independent contigs, whose relative positions and orientations along the genome being sequenced are unknown. Although several tools have been developed to order and orient the contigs of draft genomes, more accurate tools are still needed.

Results

In this study, we present a novel reference-based contig assembly (or scaffolding) tool, named as CAR, that can efficiently and more accurately order and orient the contigs of a prokaryotic draft genome based on a reference genome of a related organism. Given a set of contigs in multi-FASTA format and a reference genome in FASTA format, CAR can output a list of scaffolds, each of which is a set of ordered and oriented contigs. For validation, we have tested CAR on a real dataset composed of several prokaryotic genomes and also compared its performance with several other reference-based contig assembly tools. Consequently, our experimental results have shown that CAR indeed performs better than all these other reference-based contig assembly tools in terms of sensitivity, precision and genome coverage.

Conclusions

CAR serves as an efficient tool that can more accurately order and orient the contigs of a prokaryotic draft genome based on a reference genome. The web server of CAR is freely available at http://genome.cs.nthu.edu.tw/CAR/ and its stand-alone program can also be downloaded from the same website.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0381-3) contains supplementary material, which is available to authorized users.     

Amino acid cost and codon-usage biases in 6 prokaryotic genomes: a whole-genome analysis

For most prokaryotic organisms, amino acid biosynthesis represents a significant portion of their overall energy budget. The difference in the cost of synthesis between amino acids can be striking, differing by as much as 7-fold. Two prokaryotic organisms, Escherichia coli and Bacillus subtilis, have been shown to preferentially utilize less costly amino acids in highly expressed genes, indicating that parsimony in amino acid selection may confer a selective advantage for prokaryotes. This study confirms those findings and extends them to 4 additional prokaryotic organisms: Chlamydia trachomatis, Chlamydophila pneumoniae AR39, Synechocystis sp. PCC 6803, and Thermus thermophilus HB27. Adherence to codon-usage biases for each of these 6 organisms is inversely correlated with a coding region's average amino acid biosynthetic cost in a fashion that is independent of chemoheterotrophic, photoautotrophic, or thermophilic lifestyle. The obligate parasites C. trachomatis and C. pneumoniae AR39 are incapable of synthesizing many of the 20 common amino acids. Removing auxotrophic amino acids from consideration in these organisms does not alter the overall trend of preferential use of energetically inexpensive amino acids in highly expressed genes.     

Causes of insertion sequences abundance in prokaryotic genomes

Insertion sequences (ISs) are the smallest and most frequent transposable elements in prokaryotes where they play an important evolutionary role by promoting gene inactivation and genome plasticity. Their genomic abundance varies by several orders of magnitude for reasons largely unknown and widely speculated. The current availability of hundreds of genomes renders testable many of these hypotheses, notably that IS abundance correlates positively with the frequency of horizontal gene transfer (HGT), genome size, pathogenicity, nonobligatory ecological associations, and human association. We thus reannotated ISs in 262 prokaryotic genomes and tested these hypotheses showing that when using appropriate controls, there is no empirical basis for IS family specificity, pathogenicity, or human association to influence IS abundance or density. HGT seems necessary for the presence of ISs, but cannot alone explain the absence of ISs in more than 20% of the organisms, some of which showing high rates of HGT. Gene transfer is also not a significant determinant of the abundance of IS elements in genomes, suggesting that IS abundance is controlled at the level of transposition and ensuing natural selection and not at the level of infection. Prokaryotes engaging in obligatory associations have fewer ISs when controlled for genome size, but this may be caused by some being sexually isolated. Surprisingly, genome size is the only significant predictor of IS numbers and density. Alone, it explains over 40% of the variance of IS abundance. Because we find that genome size and IS abundance correlate negatively with minimal doubling times, we conclude that selection for rapid replication cannot account for the few ISs found in small genomes. Instead, we show evidence that IS numbers are controlled by the frequency of highly deleterious insertion targets. Indeed, IS abundance increases quickly with genome size, which is the exact inverse trend found for the density of genes under strong selection such as essential genes. Hence, for ISs, the bigger the genome the better.     

复杂基因组测序技术研究进展

复杂基因组指的是无法使用常规测序和组装手段直接解析的一类基因组,通常指包含高比例重复序列、高杂合度、极端GC含量、存在难消除异源DNA污染的基因组。为了解决复杂基因组的测序和组装问题,需要分别从基因组测序实验方法、测序技术平台、组装算法与策略3个方面进行深入研究。本文详细介绍了复杂基因组测序组装相关的现有技术与方法,并结合复杂基因组经典实例介绍了复杂基因组测序的技术解决途径和发展历程,可为制订合适的复杂基因组测序策略提供参考。     

Insertion of an intermediate repetitive sequence into a sea urchin histone-gene spacer

Summary A common polymorphism of the early embryonic histone-gene repeat ofStrongylocentrotus purpuratus is a 195-bp insertion within the H4-H2B spacer. The sequence, found as an insert in histone-gene repeats of 6 of 22 individuals screened, is also found at approximately 50 sites elsewhere in the genome of every individual. We compare the sequences of the histone-gene spacers that do and do not contain the insert. The insert is found not to have transposon-like features, and no sequence in the original spacer has been duplicated to flank the insert. There is, however, a hexanucleotide sequence that is repeated three times at one end of the insert, and the element has inserted between direct repeats of 5 bp that were present in the original spacer. One of the copies found outside the histone gene cluster was cloned and sequenced and is compared with the insert. Again, no transposon-like features are evident. Regions flanking the homologous sequence in this clone were used as hybridization probes in whole-genome blots. Results indicate that the 195-bp sequence insert is itself embedded within a larger element that is repeated within the genome. Therefore, only a portion of a larger repetitive sequence has integrated into the histone-gene spacer. The sequence features of the insert, although not typical of mobile elements, may be representative of other illegitimate recombination events.     

Sequencing and assembly of low copy and genic regions of isolated Triticum aestivum chromosome arm 7DS

The genome of bread wheat (Triticum aestivum) is predicted to be greater than 16 Gbp in size and consist predominantly of repetitive elements, making the sequencing and assembly of this genome a major challenge. We have reduced genome sequence complexity by isolating chromosome arm 7DS and applied second‐generation technology and appropriate algorithmic analysis to sequence and assemble low copy and genic regions of this chromosome arm. The assembly represents approximately 40% of the chromosome arm and all known 7DS genes. Comparison of the 7DS assembly with the sequenced genomes of rice (Oryza sativa) and Brachypodium distachyon identified large regions of conservation. The syntenic relationship between wheat, B. distachyon and O. sativa, along with available genetic mapping data, has been used to produce an annotated draft 7DS syntenic build, which is publicly available at http://www.wheatgenome.info . Our results suggest that the sequencing of isolated chromosome arms can provide valuable information of the gene content of wheat and is a step towards whole‐genome sequencing and variation discovery in this important crop.     

赖草基因组重复序列组成及染色体分布特性

【目的】赖草属植物是麦类作物遗传改良和育种重要的基因资源,但作为异源多倍体植物,关于其基因组来源仍存在较大争议。【方法】通过构建赖草属物种赖草的Cot-1DNA文库,获得大量重复序列,进一步利用荧光原位杂交技术和重复序列对赖草,以及近缘物种大赖草和祖先供体物种新麦草进行染色体荧光原位杂交涂染。【结果】（1）根据序列及基因组分布特性,赖草Cot-1DNA可归为串联重复序列（TaiI、Lt1-6、pTa535和pSc250家族）,散布重复序列（LTR/Gypsy、LTR/Copia、LTR及转座子）,散布加串联混合重复序列（LTR+Afa-family和N8-family+LZ-NBS-LRR）以及未能鉴定类型,4种类型在Cot-1DNA文库克隆中的占比分别为32.4%、45.7%、12.4%和9.5%。（2）串联重复序列TaiI、Lt1-6、pTa535和pSc250在不同物种及同一物种不同材料间信号数量存在较大变异,分别为7~20、1~14、17~26及0~24。（3）10个反转座子序列在所有物种染色体的分布呈现3种方式：第1种是在所有染色体上杂交信号集中分布在着丝粒、近着丝粒及间质区;第2种是在所有染色体的所有区域都有分布;第3种为大部分染色体上的分布方式与第1种相同,但是部分染色体端部也有分布。2个LTR/Copia序列仅在赖草染色体上有分布,其他序列在不同物种以及不同材料间均有分布,但是在信号强度以及部分染色体上的分布方式等存在多态性。【结论】赖草属物种中的一些重复序列可能具有快速进化的特性,支持赖草属物种多倍化过程中,可能存在散在重复序列向整个核基因组的快速同质化扩散。     

Survey for g-proteins in the prokaryotic genomes: prediction of functional roles based on classification

The members of the family of G-proteins are characterized by their ability to bind and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). Despite a common biochemical function of GTP hydrolysis shared among the members of the family of G-proteins, they are associated with diverse biological roles. The current work describes the identification and detailed analysis of the putative G-proteins encoded in the completely sequenced prokaryotic genomes. Inferences on the biological roles of these G-proteins have been obtained by their classification into known functional subfamilies. We have identified 497 G-proteins in 42 genomes. Seven small GTP-binding protein homologues have been identified in prokaryotes with at least two of the diagnostic sequence motifs of G-proteins conserved. The translation factors have the largest representation (234 sequences) and are found to be ubiquitous, which is consistent with their critical role in protein synthesis. The GTP_OBG subfamily comprises of 79 sequences in our dataset. A total of 177 sequences belong to the subfamily of GTPase of unknown function and 154 of these could be associated with domains of known functions such as cell cycle regulation and t-RNA modification. The large GTP-binding proteins and the alpha-subunit of heterotrimeric G-proteins are not detected in the genomes of the prokaryotes surveyed.     

Identification and categorization of horizontally transferred genes in prokaryotic genomes

Horizontal gene transfer （HGT）, a process through which genomes acquire genetic materials from distantly related organisms, is believed to be one of the major forces in prokaryotic genome evolution.However, systematic investigation is still scarce to clarify two basic issues about HGT： （1） what types of genes are transferred; and （2） what influence HGT events over the organization and evolution of biological pathways. Genome-scale investigations of these two issues will advance the systematical understanding of HGT in the context of prokaryotic genome evolution. Having investigated 82 genomes, we constructed an HGT database across broad evolutionary timescales. We identified four function categories containing a high proportion of horizontally transferred genes： cell envelope, energy metabolism, regulatory functions, and transport/binding proteins. Such biased function distribution indicates that HGT is not completely random;instead, it is under high selective pressure, required by function restraints in organisms. Furthermore, we mapped the transferred genes onto the connectivity structure map of organism-specific pathways listed in Kyoto Encyclopedia of Genes and Genomes （KEGG）. Our results suggest that recruitment of transferred genes into pathways is also selectively constrained because of the tuned interaction between original pathway members. Pathway organization structures still conserve well through evolution even with the recruitment of horizontally transferred genes. Interestingly, in pathways whose organization were significantly affected by HGT events, the operon-like arrangement of transferred genes was found to be prevalent. Such results suggest that operon plays an essential and directional role in the integration of alien genes into pathways.     

Physical mapping of repetitive sequences and genome analysis in six Elymus species by in situ hybridization

Genome constitution and genetic relationships between six Elymus species were assessed by physical mapping of different repetitive sequences using a technique of sequential fluorescence in situ hybridization and genomic in situ hybridization.The six Elymus species are all naturally growing species in northwest China,namely,E.sibiricus,E.nutans,E.barystachyus,E.xiningensis,E.excelsus,and E.dahuricus.An StStHH genome constitution was revealed for E.sibiricus and StStHHYY for the remainder species.Each chromosome could be clearly characterized by physical mapping with 18S-26S rDNA,5S rDNA,Afa-family,and AAG repeats,and be allocated to a certain genome by genomic in situ hybridization.Two 5S rDNA sites,each in the H and St genomes,and three 18S-26S rDNA sites,two in the St genome and one in the Y genome,were uncovered in most of the species.The strong Afa-family hybridization signals discriminated the H genome from the St and Y genomes.The H and Y genome carried more AAG repeats than St.A common non-Robertsonian reciprocal translocation between the H and Y genomes was revealed in E.barystachyus,E.xiningensis,E.excelsus and E.dahuricus.Comparison of molecular karyotypes strongly suggests that they can be classified into three groups,namely,E.sibiricus,E.nutans,and others.     

Quantitation of Repetitive Sequences in Human Genomic DNA and Detection of an Elevated Ribosomal Repeat Copy Number in Schizophrenia: The Results of Molecular and Cytogenetic Analyses

A modified version of quantitating repetitive sequences in genomic DNA was developed to allow comparisons for numerous individual genomes and simultaneous analysis of several sequences in each DNA specimen. The relative genomic content of ribosomal repeats (rDNA) was estimated for 75 individuals, including 33 healthy donors (HD) and 42 schizophrenic patients (SP). The rDNA copy number in HD was 427 ± 18 (mean ± SE) per diploid nucleus, ranging 250–600. In SP, the rDNA copy number was 494 ± 15 and ranged 280–670, being significantly higher than in HD. The two samples did not differ in contents of sequences hybridizing with probes directed to a subfraction of human satellite III or to the histone genes. Cytogenetic analysis (silver staining of metaphase chromosomes) showed that the content of active rRNA genes in nucleolus organizer regions is higher in SP compared with HD. The possible causes of the elevated rRNA gene dosage in SP were considered. The method employed was proposed for studying the polymorphism for genomic content of various repeats in higher organisms, including humans.     

Identification of a dispersed MboI repeat family in five higher plant genomes

Digestion of nuclear DNAs of five plants, namelyCucurbita maxima (red gourd),Trichosanthes anguina (snake gourd),Cucumis sativus (cucumber),Cajanus cajan (pigeon pea) andPhaseolus vulgaris (french bean) with the restriction endonucleaseMboI yielded discrete size classes with molecular weights in the range of 0.5 to 5 kbp. TheMboI digestion pattern of Cot 0.1 DNA in french bean is comparable with that of total DNA, indicating that these bands represented highly repeated DNA sequences. Cleavage of the DNAs with varying amounts ofMboI indicated the dispersed nature of the repeat families. Southern hybridization studies using french bean highly repetitive DNA as a probe indicated more homology with repeats of pigeon pea and less homology with red gourd, snake gourd and cucumber repeats.     

Multiple forms of male-specific simple repetitive sequences in the genusMus

Summary Previous reports indicate that in laboratory strains of mice, males are distinct from females in possession of repetitive DNA, notably devoid of Eco RI and Hae III sites and rich in the simple tetranucleotides GATA/GACA. We report here that such sequences originated in an ancestor common to laboratory mice,Mus hortulanus, M. spretus, and possibly alsoM. cookii. Interestingly, other male-specific satellite sequences were detected inM. caroli, M. cookii, M. saxicola, andM. minutoides. This novel satellite is also likely to be composed of simple repetitious sequences, but does not contain GATA and GACA. Thus, the Y chromosome appears to contain a disproportionately large amount of simple repetitious DNA. An attractive explanation for these results is that long tandem arrays of simple repeated sequences are generated at high frequency throughout the genome and that they are retained for a longer time on the Y chromosome due to the absence of homologous pairing at meiosis.     

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

In prokaryotic cells, 3′–5′ exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3′–5′ untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base “N.” Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3′-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3′-terminus of the mRNA by blocking the processing action of the 3′–5′ exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.     

第二、三代基因组测序数据混合拼接软件综述

DNA测序是生物信息学研究的重要内容之一,对测序序列的从头拼接是其中非常基础而重要的步骤.随着测序技术的不断更新,新的第三代测序数据拥有更长的序列长度、高错误率等性质,针对这些性质,同时使用二代、三代测序数据进行混合拼接是获得更好的拼接结果一种重要方式.本文介绍了现有的混合拼接软件的基本原理,并比较了不同软件拼接结果....     

Junker: an intergenic explorer for bacterial genomes

In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those "junk" regions for genomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant plots, GC percentage and repeat details. Upon selection of a particular bacterial genome, the physical genome map is displayed as a multiple loci with options to view any loci of interest in detail. In addition, an IGR statistics module has been created and implemented in the web server to analyze the length distribution of the IGRs and to understand the disordered grouping of IGRs across the genome by generating the four-quadrant plots. The proposed web server is freely available at the URL http://pranag.physics.iisc.ernet.in/junker/.     

Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens

Whole‐genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in‐depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project‐specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all‐purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real‐world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.