Functional analysis of the yeast genome

Just as Saccharomyces cerevisiae itself provides a model for so many processes essential to eukaryotic life, we anticipate that the methods and the mindset that have moved yeast biological research "beyond the genome" provide a prototype for making similar progress in other organisms. In this review I describe the experimental processes, results and utility of the current large-scale experimental approaches that use genomic data to provide a functional analysis of the yeast genome. Electronic Publication     

Isochores,GC3 and mutation biases in the human genome

In this work we re-examined the hypothesis that the variation in GC content in the human genome is due to different regional mutational biases. For this purpose we inferred the mutational pattern by using mutation databases that are available for many genes associated with human genetic diseases. The assumption of this approach is that such mutations reflect the actual frequency distribution of mutations as they arise in the population. Four classes of genes, classified according to their GC₃ level, were included in this study: GC₃-poor genes (GC₃<45%), genes with intermediate GC₃ content (45%3<60%), GC₃-rich genes (60%3<75%) and very GC₃-rich genes (GC₃>75%). Our results show that most genes are under AT mutational biases, with very little variation compared to the expectations of neutral GC level. It is noteworthy that the mutational patterns in the GC₃-rich genes do not appear to account for their GC₃-richness. Instead, GC₃-rich and very GC₃-rich genes exhibit patterns of mutations that yield expectations of neutral GC₃ content that are much lower than their actual GC₃.     

The distribution of surviving blocks of an ancestral genome

What is the chance that some part of a stretch of genome will survive? In a population of constant size, and with no selection, the probability of survival of some part of a stretch of map length y < 1 approaches y/log(yt/2) for log(yt) > or = 1. Thus, the whole genome is certain to be lost, but the rate of loss is extremely slow. This solution extends to give the whole distribution of surviving block sizes as a function of time. We show that the expected number of blocks at time t is 1+yt and give expressions for the moments of the number of blocks and the total amount of genome that survives for a given time. The solution is based on a branching process and assumes complete interference between crossovers, so that each descendant carries only a single block of ancestral material. We consider cases where most individuals carry multiple blocks, either because there are multiple crossovers in a long genetic map, or because enough time has passed that most individuals in the population are related to each other. For species such as ours, which have a long genetic map, the genome of any individual which leaves descendants (approximately 80% of the population for a Poisson offspring number with mean two) is likely to persist for an extremely long time, in the form of a few short blocks of genome.     

The promise of a protist: the Dictyostelium genome project

The genome of Dictyostelium discoideum is being sequenced by an international consortium and is scheduled for completion in the next few years. The sequence will accelerate research into a number of phenomena carried out by these versatile soil amoebae, providing insight into analogous processes that operate in a wide range of eukaryotes. These include the dynamic regulation of the cytoskeleton during chemotaxis, intercellular communication during multicellular development and the intracellular growth of bacterial pathogens. The current state of the genome project is summarized and the challenges of sequencing a genome with unusually low guanine and cytosine content and with a bimodal base composition distribution are discussed. The prospects for functional analyses at the genomic scale are also considered. Electronic Publication     

HLA-E, HLA-F, and HLA-G polymorphism: genomic sequence defines haplotype structure and variation spanning the nonclassical class I genes

Despite several studies that defined the polymorphism of the nonclassical human leukocyte antigen-E (HLA-E), HLA-F, and HLA-G genes, most polymorphisms thus far examined in correlative studies were derived from the coding sequences of these genes. In addition, some discrepancies and ambiguities in the available data have persisted in current databases. To expand the data available and to resolve some of the discrepant data, we have defined protocols that allow for the amplification of 6 to 7 kb of contiguous genomic sequence for each gene, including all of the coding and intron sequences, approximately 2 kb of 5' flanking promoter sequence, and 1 kb of 3' flanking sequence. Using long-range polymerase chain reaction (PCR) protocols, generating either one or two PCR products depending on the locus, amplified genomic DNA was directly sequenced to completion using a set of about 30 primers over each locus to yield contiguous sequence data from both strands. Using this approach, we sequenced 33 genomic DNAs, from Asian, African American, and Caucasian samples. The results of this analysis confirmed several previously reported coding sequence variants, identified several new allelic variants, and also defined extensive variation in intron and flanking sequences. It was possible to construct haplotype maps and to identify tagging single nucleotide polymorphisms that can be used to detect the composite variation spanning all three genes.     

Looking back, looking beyond: revisiting the ethics of genome generation

This paper will explore some of the ethical imperatives that have shaped strategic and policy frameworks for the use of new genetic technologies and how these play a role in shaping the nature of research and changing attitudes; with an attempt to conceptualize some theories of genetic determinism. I analyse why there is a need to put bioethical principles within a theoretical framework in the context of new technologies, and how, by doing so, their practical applications for agriculture, environment medicine and health care can be legitimized. There are several theories in favour of and against the use of genetic technologies that focus on genes and their role in our existence. In particular the theory of geneticisation is commonly debated. It highlights the conflicting interests of science, society and industry in harnessing genetic knowledge when the use of such knowledge could challenge ethical principles. Critics call it a ‘reductionist’ approach, based on arguments that are narrowed down to genes, often ignoring other factors including biological, social and moral ones. A parallel theory is that there is something special about genes, and it is this “genetic exceptionalism” that creates hopes and myths. Either way, the challenging task is to develop a common ground for understanding the importance of ethical sensitivities. As research agendas become more complex, ethical paradigms will need to be more influential. New principles are needed to answer the complexities of ethical issues as complex technologies develop. This paper reflects on global ethical principles and the tensions between ethical principles in legitimizing genetic technologies at the social and governance level.     

Evaluating the accuracy of SHAPE-directed RNA secondary structure predictions

Recent advances in RNA structure determination include using data from high-throughput probing experiments to improve thermodynamic prediction accuracy. We evaluate the extent and nature of improvements in data-directed predictions for a diverse set of 16S/18S ribosomal sequences using a stochastic model of experimental SHAPE data. The average accuracy for 1000 data-directed predictions always improves over the original minimum free energy (MFE) structure. However, the amount of improvement varies with the sequence, exhibiting a correlation with MFE accuracy. Further analysis of this correlation shows that accurate MFE base pairs are typically preserved in a data-directed prediction, whereas inaccurate ones are not. Thus, the positive predictive value of common base pairs is consistently higher than the directed prediction accuracy. Finally, we confirm sequence dependencies in the directability of thermodynamic predictions and investigate the potential for greater accuracy improvements in the worst performing test sequence.     

Tick genomics: the Ixodes genome project and beyond

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.     

Begin at the beginning: A BAC-end view of the passion fruit (Passiflora) genome

Background

The passion fruit (Passiflora edulis) is a tropical crop of economic importance both for juice production and consumption as fresh fruit. The juice is also used in concentrate blends that are consumed worldwide. However, very little is known about the genome of the species. Therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. In this study, we have constructed a large-insert BAC library and provided the first view on the structure and content of the passion fruit genome, using BAC-end sequence (BES) data as a major resource.

Results

The library consisted of 82,944 clones and its levels of organellar DNA were very low. The library represents six haploid genome equivalents, and the average insert size was 108 kb. To check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight Passiflora gene sequences available in public databases. BACs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four BACs in three chromosomes (n = 9). Then, we explored 10,000 BES and we identified reads likely to contain repetitive mobile elements (19.6% of all BES), simple sequence repeats and putative proteins, and to estimate the GC content (~42%) of the reads. Around 9.6% of all BES were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed (940). The vast majority of the top-hits made by our sequences were to Populus trichocarpa (24.8% of the total occurrences), Theobroma cacao (21.6%), Ricinus communis (14.3%), Vitis vinifera (6.5%) and Prunus persica (3.8%).

Conclusions

We generated the first large-insert library for a member of Passifloraceae. This BAC library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. Remarkably, a number of BAC-end pair sequences could be mapped to intervals of the sequenced Arabidopsis thaliana, V. vinifera and P. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-816) contains supplementary material, which is available to authorized users.     

MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome

Here we present MethylQuant, a novel method that allows accurate quantification of the methylation level of a specific cytosine within a complex genome. This method relies on the well-established treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil without modifying 5-methyl cytosine. The region of interest is then PCR-amplified and quantification of the methylation status of a specific cytosine is performed by methylation-specific real-time PCR with SYBR Green I using one of the primers whose 3′ end discriminates between the methylation states of this cytosine. The presence of a locked nucleic acid at the 3′ end of the discriminative primer provides the specificity necessary for accurate and sensitive quantification, even when one of the methylation states is present at a level as low as 1% of the overall population. We demonstrate that accurate quantification of the methylation status of specific cytosines can be achieved in biological samples. The method is high-throughput, cost-effective, relatively simple and does not require any specific equipment other than a real-time PCR instrument.     

Sequence and analysis of a whole genome from Kuwaiti population subgroup of Persian ancestry

Background

The 1000 Genome project paved the way for sequencing diverse human populations. New genome projects are being established to sequence underrepresented populations helping in understanding human genetic diversity. The Kuwait Genome Project an initiative to sequence individual genomes from the three subgroups of Kuwaiti population namely, Saudi Arabian tribe; “tent-dwelling” Bedouin; and Persian, attributing their ancestry to different regions in Arabian Peninsula and to modern-day Iran (West Asia). These subgroups were in line with settlement history and are confirmed by genetic studies. In this work, we report whole genome sequence of a Kuwaiti native from Persian subgroup at >37X coverage.

Results

We document 3,573,824 SNPs, 404,090 insertions/deletions, and 11,138 structural variations. Out of the reported SNPs and indels, 85,939 are novel. We identify 295 ‘loss-of-function’ and 2,314 ’deleterious’ coding variants, some of which carry homozygous genotypes in the sequenced genome; the associated phenotypes include pharmacogenomic traits such as greater triglyceride lowering ability with fenofibrate treatment, and requirement of high warfarin dosage to elicit anticoagulation response. 6,328 non-coding SNPs associate with 811 phenotype traits: in congruence with medical history of the participant for Type 2 diabetes and β-Thalassemia, and of participant’s family for migraine, 72 (of 159 known) Type 2 diabetes, 3 (of 4) β-Thalassemia, and 76 (of 169) migraine variants are seen in the genome. Intergenome comparisons based on shared disease-causing variants, positions the sequenced genome between Asian and European genomes in congruence with geographical location of the region. On comparison, bead arrays perform better than sequencing platforms in correctly calling genotypes in low-coverage sequenced genome regions however in the event of novel SNP or indel near genotype calling position can lead to false calls using bead arrays.

Conclusions

We report, for the first time, reference genome resource for the population of Persian ancestry. The resource provides a starting point for designing large-scale genetic studies in Peninsula including Kuwait, and Persian population. Such efforts on populations under-represented in global genome variation surveys help augment current knowledge on human genome diversity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1233-x) contains supplementary material, which is available to authorized users.     

The whole mitochondrial genome sequence of the zebra finch (Taeniopygia guttata)

Here we describe the complete nucleotide sequence of the mitochondrial genome (16 583/4 bp) of the zebra finch (Taeniopygia guttata). Primers were designed based on highly conserved regions of an alignment of three passerine complete mitochondrial DNA (mtDNA) sequences. A combination of overlapping long polymerase chain reaction (PCR) purification, followed by fully nested PCR and sequencing was used to determine the complete mtDNA genome. Six birds, from distinct maternal lineages of a pedigreed population were sequenced. Five novel haplotypes were identified. These sequences provide the first data for sequence variation across the whole mitochondrial genome of a passerine bird species.     

EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome

In a panel of seven genotypes, 437 expressed sequence tag (EST)-derived DNA fragments were sequenced. Single nucleotide polymorphisms (SNPs) that were polymorphic between the parents of three mapping populations were mapped by heteroduplex analysis and a genome-wide consensus map comprising 216 EST-derived SNPs and 4 InDel (insertion/deletion) markers was constructed. The average frequency of SNPs amounted to 1/130 bp and 1/107.8 bp for a set of randomly selected and a set of mapped ESTs, respectively. The calculated nucleotide diversities (π) ranged from 0 to 40.0 × 10⁻³ (average 3.1 × 10⁻³) and 0.52 × 10⁻³ to 39.51 × 10^–3 (average 4.37 × 10⁻³) for random and mapped ESTs, respectively. The polymorphism information content value for mapped SNPs ranged from 0.24 to 0.50 with an average of 0.34. As expected, combination of SNPs present in an amplicon (haplotype) exhibited a higher information content ranging from 0.24 to 0.85 with an average of 0.50. Cleaved amplified polymorphic sequence assays (including InDels) were designed for a total of 87 (39.5%) SNP markers. The high abundance of SNPs in the barley genome provides avenues for the systematic development of saturated genetic maps and their integration with physical maps. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Both R. Kota and R.K. Varshney contributed equally to this work.     

Evaluation of genome skimming to detect and characterise human and livestock helminths

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.     

Steady progress and recent breakthroughs in the accuracy of automated genome annotation

The sequencing of large, complex genomes has become routine, but understanding how sequences relate to biological function is less straightforward. Although much attention is focused on how to annotate genomic features such as developmental enhancers and non-coding RNAs, there is still no higher eukaryote for which we know the correct exon-intron structure of at least one ORF for each gene. Despite this uncomfortable truth, genome annotation has made remarkable progress since the first drafts of the human genome were analysed. By combining several computational and experimental methods, we are now closer to producing complete and accurate gene catalogues than ever before.     

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.     

Evaluating the accuracy of uterine cancer screening with the regional cancer registration system

OBJECTIVE: To evaluate the effectiveness of uterine cancer screening by analyzing the accuracy of cervical and endometrial cytodiagnoses as screening methods. STUDY DESIGN: During the year of April 1, 1991-March 31, 1992, 186,161 and 5,697 women underwent cervical and endometrial cytodiagnoses, respectively, and their cytodiagnostic results were computer registered at the Miyagi Cancer Society. By comparison of these examinees with 753 cancer patients who were registered at the regional cancer registry between 1991 and 1993, 133 individuals who were assumed to be identical between the two systems were selected, and of these cases, 83 patients, including test-positive cases, were found within one year. The sensitivity and specificity of each screening method were investigated. RESULTS: Regarding examinees diagnosed as having cancer by the same month in the following year after diagnosis on screening as false negative, the sensitivity, specificity and false negative rates of cervical cytodiagnosis were 94.7%, 98.9% and 5.3%, respectively, and those of endometrial cytodiagnosis were 83.3%, 96.7% and 16.7%, respectively. CONCLUSION: In comparison with the accuracy of cancer examinations for other organs performed by the health care administration, the accuracy of cervical and endometrial cytodiagnoses was sufficient to designate them screening methods.     

Putative lipoproteins of Streptococcus agalactiae identified by bioinformatic genome analysis

Streptococcus agalactiae is a significant pathogen causing invasive disease in neonates and thus an understanding of the molecular basis of the pathogenicity of this organism is of importance. N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes. Lipidation is directed by the presence of a cysteine-containing 'lipobox' within specific signal peptides and this feature has greatly facilitated the bioinformatic identification of putative lipoproteins. We have designed previously a taxon-specific pattern (G+LPP) for the identification of Gram-positive bacterial lipoproteins, based on the signal peptides of experimentally verified lipoproteins (Sutcliffe I.C. and Harrington D.J. Microbiology 148: 2065-2077). Patterns searches with this pattern and other bioinformatic methods have been used to identify putative lipoproteins in the recently published genomes of S. agalactiae strains 2603/V and NEM316. A core of 39 common putative lipoproteins was identified, along with 5 putative lipoproteins unique to strain 2603/V and 2 putative lipoproteins unique to strain NEM316. Thus putative lipoproteins represent ca. 2% of the S. agalactiae proteome. As in other Gram-positive bacteria, the largest functional category of S. agalactiae lipoproteins is that predicted to comprise of substrate binding proteins of ABC transport systems. Other roles include lipoproteins that appear to participate in adhesion (including the previously characterised Lmb protein), protein export and folding, enzymes and several species-specific proteins of unknown function. These data suggest lipoproteins may have significant roles that influence the virulence of this important pathogen.