Sequencing and Analysis of Globally Obtained Human Respiratory Syncytial Virus A and B Genomes

Background

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level.

Methods

We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference.

Results

We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10^-4 substitutions/site/year (95% HPD 5.61 × 10^-4 to 7.6 × 10^-4) and for RSVB the evolutionary rate was 7.69 × 10^-4 substitutions/site/year (95% HPD 6.81 × 10^-4 to 8.62 × 10^-4). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB.

Conclusions

Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.     

Using the taxon-specific genes for the taxonomic classification of bacterial genomes

Background

The correct taxonomic assignment of bacterial genomes is a primary and challenging task. With the availability of whole genome sequences, the gene content based approaches appear promising in inferring the bacterial taxonomy. The complete genome sequencing of a bacterial genome often reveals a substantial number of unique genes present only in that genome which can be used for its taxonomic classification.

Results

In this study, we have proposed a comprehensive method which uses the taxon-specific genes for the correct taxonomic assignment of existing and new bacterial genomes. The taxon-specific genes identified at each taxonomic rank have been successfully used for the taxonomic classification of 2,342 genomes present in the NCBI genomes, 36 newly sequenced genomes, and 17 genomes for which the complete taxonomy is not yet known. This approach has been implemented for the development of a tool ‘Microtaxi’ which can be used for the taxonomic assignment of complete bacterial genomes.

Conclusion

The taxon-specific gene based approach provides an alternate valuable methodology to carry out the taxonomic classification of newly sequenced or existing bacterial genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1542-0) contains supplementary material, which is available to authorized users.     

Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

Background

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.

Results

We developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.

Conclusions

The P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-737) contains supplementary material, which is available to authorized users.     

Whole Genome Sequence of the Treponema pallidum subsp. endemicum Strain Bosnia A: The Genome Is Related to Yaws Treponemes but Contains Few Loci Similar to Syphilis Treponemes

Background

T. pallidum subsp. endemicum (TEN) is the causative agent of bejel (also known as endemic syphilis). Clinical symptoms of syphilis and bejel are overlapping and the epidemiological context is important for correct diagnosis of both diseases. In contrast to syphilis, caused by T. pallidum subsp. pallidum (TPA), TEN infections are usually spread by direct contact or contaminated utensils rather than by sexual contact. Bejel is most often seen in western Africa and in the Middle East. The strain Bosnia A was isolated in 1950 in Bosnia, southern Europe.

Methodology/Principal Findings

The complete genome of the Bosnia A strain was amplified and sequenced using the pooled segment genome sequencing (PSGS) method and a combination of three next-generation sequencing techniques (SOLiD, Roche 454, and Illumina). Using this approach, a total combined average genome coverage of 513× was achieved. The size of the Bosnia A genome was found to be 1,137,653 bp, i.e. 1.6–2.8 kbp shorter than any previously published genomes of uncultivable pathogenic treponemes. Conserved gene synteny was found in the Bosnia A genome compared to other sequenced syphilis and yaws treponemes. The TEN Bosnia A genome was distinct but very similar to the genome of yaws-causing T. pallidum subsp. pertenue (TPE) strains. Interestingly, the TEN Bosnia A genome was found to contain several sequences, which so far, have been uniquely identified only in syphilis treponemes.

Conclusions/Significance

The genome of TEN Bosnia A contains several sequences thought to be unique to TPA strains; these sequences very likely represent remnants of recombination events during the evolution of TEN treponemes. This finding emphasizes a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.     

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.     

Full genome SNP-based phylogenetic analysis reveals the origin and global spread of Brucella melitensis

Background

Brucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis.

Results

Single sequencing runs of each genome resulted in draft genome sequences of MY1483/09 and Phil1136/12, which covered 99.85% and 99.92% of the complete genome sequences, respectively. The B. melitensis genome sequences, and two B. abortus strains used as the outgroup strains, yielded a total of 13,728 SNP sites. Phylogenetic analysis using whole-genome SNPs and geographical distribution of the isolates revealed spatial clustering of the B. melitensis isolates into five genotypes, I, II, III, IV and V. The Mediterranean strains, identified as genotype I, occupied the basal node of the phylogenetic tree, suggesting that B. melitensis may have originated from the Mediterranean regions. All of the Asian B. melitensis strains clustered into genotype II with the SEA strains, including the two isolates sequenced in this study, forming a distinct clade denoted here as genotype IId. Genotypes III, IV and V of B. melitensis demonstrated a restricted geographical distribution, with genotype III representing the African lineage, genotype IV representing the European lineage and genotype V representing the American lineage.

Conclusion

We showed that SNPs retrieved from the B. melitensis draft full genomes were sufficient to resolve the interspecies relationships between B. melitensis strains and to discriminate between the vaccine and endemic strains. Phylogeographic reconstruction of the history of B. melitensis global spread at a finer scale by using whole-genome SNP analyses supported the origin of all B. melitensis strains from the Mediterranean region. The possible global distribution of B. melitensis following the ancient trade routes was also consistent with whole-genome SNP phylogeny. The whole genome SNP phylogenetics analysis, hence is a powerful tool for intraspecies discrimination of closely related species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1294-x) contains supplementary material, which is available to authorized users.     

Comparative genomics of first available bovine Anaplasma phagocytophilum genome obtained with targeted sequence capture

Background

Anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. As A. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain.Our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with A. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains.

Results

DNA was extracted from a bovine blood sample infected by A. phagocytophilum. Following a whole genome capture approach, A. phagocytophilum DNA was enriched 197-fold in the sample and then sequenced using Illumina technology. In total, 58.9% of obtained reads corresponded to the A. phagocytophilum genome, covering 85.3% of the HZ genome. Then by performing comparisons with nine previously-sequenced A. phagocytophilum genomes, we determined the core genome of these ten strains. Following analysis, 1281 coding DNA sequences, including 1001 complete sequences, were detected in the A. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. These four coding DNA sequences coded for "hypothetical proteins of unknown function” and require further analysis. We also identified nine proteins common to both European domestic ruminants tested.

Conclusion

Using a whole genome capture approach, we have sequenced the first A. phagocytophilum genome isolated from a cow. To the best of our knowledge, this is the first time that this method has been used to selectively enrich pathogenic bacterial DNA from samples also containing host DNA. The four proteins unique to the A. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-973) contains supplementary material, which is available to authorized users.     

The Fast Changing Landscape of Sequencing Technologies and Their Impact on Microbial Genome Assemblies and Annotation

Background

The emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation.

Methodology/Principal Findings

In this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis.

Conclusion

These data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).     

Reducing assembly complexity of microbial genomes with single-molecule sequencing

Background

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.

Results

To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.

Conclusions

Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.     

Genome sequence of ground tit Pseudopodoces humilis and its adaptation to high altitude

Background

The mechanism of high-altitude adaptation has been studied in certain mammals. However, in avian species like the ground tit Pseudopodoces humilis, the adaptation mechanism remains unclear. The phylogeny of the ground tit is also controversial.

Results

Using next generation sequencing technology, we generated and assembled a draft genome sequence of the ground tit. The assembly contained 1.04 Gb of sequence that covered 95.4% of the whole genome and had higher N50 values, at the level of both scaffolds and contigs, than other sequenced avian genomes. About 1.7 million SNPs were detected, 16,998 protein-coding genes were predicted and 7% of the genome was identified as repeat sequences. Comparisons between the ground tit genome and other avian genomes revealed a conserved genome structure and confirmed the phylogeny of ground tit as not belonging to the Corvidae family. Gene family expansion and positively selected gene analysis revealed genes that were related to cardiac function. Our findings contribute to our understanding of the adaptation of this species to extreme environmental living conditions.

Conclusions

Our data and analysis contribute to the study of avian evolutionary history and provide new insights into the adaptation mechanisms to extreme conditions in animals.     

Whole-genome synthesis and characterization of viable S13-like bacteriophages

Background

Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.

Methodology/Principal Findings

The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl₂, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.

Conclusions/Significance

The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.     

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome

Background and Aims

Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes.

Methods

The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues.

Key Results

BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity.

Conclusions

A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.     

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Background

In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results

Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions

The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1476-6) contains supplementary material, which is available to authorized users.     

The Complete Mitochondrial Genome of Gossypium hirsutum and Evolutionary Analysis of Higher Plant Mitochondrial Genomes

Background

Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes.

Methodology/Principal Findings

We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes.

Conclusion

The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.     

Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence

Background

The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents.

Methodology/Principal Findings

To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (d_A) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function.

Conclusions/Significance

Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics.     

High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis

Background

There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome.

Results

The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full- sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage.

Conclusions

The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-713) contains supplementary material, which is available to authorized users.     

Whole genome resequencing in tomato reveals variation associated with introgression and breeding events

Background

One of the goals of genomics is to identify the genetic loci responsible for variation in phenotypic traits. The completion of the tomato genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of genetic variation present in the tomato genome. Like many self-pollinated crops, cultivated tomato accessions show a low molecular but high phenotypic diversity. Here we describe the whole-genome resequencing of eight accessions (four cherry-type and four large fruited lines) chosen to represent a large range of intra-specific variability and the identification and annotation of novel polymorphisms.

Results

The eight genomes were sequenced using the GAII Illumina platform. Comparison of the sequences with the reference genome yielded more than 4 million single nucleotide polymorphisms (SNPs). This number varied from 80,000 to 1.5 million according to the accessions. Almost 128,000 InDels were detected. The distribution of SNPs and InDels across and within chromosomes was highly heterogeneous revealing introgressions from wild species and the mosaic structure of the genomes of the cherry tomato accessions. In-depth annotation of the polymorphisms identified more than 16,000 unique non-synonymous SNPs. In addition 1,686 putative copy-number variations (CNVs) were identified.

Conclusions

This study represents the first whole genome resequencing experiment in cultivated tomato. Substantial genetic differences exist between the sequenced tomato accessions and the reference sequence. The heterogeneous distribution of the polymorphisms may be related to introgressions that occurred during domestication or breeding. The annotated SNPs, InDels and CNVs identified in this resequencing study will serve as useful genetic tools, and as candidate polymorphisms in the search for phenotype-altering DNA variations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-791) contains supplementary material, which is available to authorized users.     

A Genome-Wide Analysis of Genetic Diversity in Trypanosoma cruzi Intergenic Regions

Background

Trypanosoma cruzi is the causal agent of Chagas Disease. Recently, the genomes of representative strains from two major evolutionary lineages were sequenced, allowing the construction of a detailed genetic diversity map for this important parasite. However this map is focused on coding regions of the genome, leaving a vast space of regulatory regions uncharacterized in terms of their evolutionary conservation and/or divergence.

Methodology

Using data from the hybrid CL Brener and Sylvio X10 genomes (from the TcVI and TcI Discrete Typing Units, respectively), we identified intergenic regions that share a common evolutionary ancestry, and are present in both CL Brener haplotypes (TcII-like and TcIII-like) and in the TcI genome; as well as intergenic regions that were conserved in only two of the three genomes/haplotypes analyzed. The genetic diversity in these regions was characterized in terms of the accumulation of indels and nucleotide changes.

Principal Findings

Based on this analysis we have identified i) a core of highly conserved intergenic regions, which remained essentially unchanged in independently evolving lineages; ii) intergenic regions that show high diversity in spite of still retaining their corresponding upstream and downstream coding sequences; iii) a number of defined sequence motifs that are shared by a number of unrelated intergenic regions. A fraction of indels explains the diversification of some intergenic regions by the expansion/contraction of microsatellite-like repeats.