Lars Bolund

WEGO: a web tool for plotting GO annotations

Unified, structured vocabularies and classifications freely provided by the Gene Ontology (GO) Consortium are widely accepted in most of the large scale gene annotation projects. Consequently, many tools have been created for use with the GO ontologies. WEGO (Web Gene Ontology Annotation Plot) is a simple but useful tool for visualizing, comparing and plotting GO annotation results. Different from other commercial software for creating chart, WEGO is designed to deal with the directed acyclic graph structure of GO to facilitate histogram creation of GO annotation results. WEGO has been used widely in many important biological research projects, such as the rice genome project and the silkworm genome project. It has become one of the daily tools for downstream gene annotation analysis, especially when performing comparative genomics tasks. WEGO, along with the two other tools, namely External to GO Query and GO Archive Query, are freely available for all users at . There are two available mirror sites at and . Any suggestions are welcome at wego@genomics.org.cn.

The diploid genome sequence of an Asian individual

Here we present the first diploid genome sequence of an Asian individual. The genome was sequenced to 36-fold average coverage using massively parallel sequencing technology. We aligned the short reads onto the NCBI human reference genome to 99.97% coverage, and guided by the reference genome, we used uniquely mapped reads to assemble a high-quality consensus sequence for 92% of the Asian individual’s genome. We identified approximately 3 million single-nucleotide polymorphisms (SNPs) inside this region, of which 13.6% were not in the dbSNP database. Genotyping analysis showed that SNP identification had high accuracy and consistency, indicating the high sequence quality of this assembly. We also carried out heterozygote phasing and haplotype prediction against HapMap CHB and JPT haplotypes (Chinese and Japanese, respectively), sequence comparison with the two available individual genomes (J. D. Watson and J. C. Venter), and structural variation identification. These variations were considered for their potential biological impact. Our sequence data and analyses demonstrate the potential usefulness of next-generation sequencing technologies for personal genomics.

Sequencing of 50 Human Exomes Reveals Adaptation to High Altitude

No Genetic Vertigo Peoples living in high altitudes have adapted to their situation (see the Perspective by Storz). To identify gene regions that might have contributed to high-altitude adaptation in Tibetans, Simonson et al. (p. 72, published online 13 May) conducted a genome scan of nucleotide polymorphism comparing Tibetans, Han Chinese, and Japanese, while Yi et al. (p. 75) performed comparable analyses on the coding regions of all genes—their exomes. Both studies converged on a gene, endothelial Per-Arnt-Sim domain protein 1 (also known as hypoxia-inducible factor 2α), which has been linked to the regulation of red blood cell production. Other genes identified that were potentially under selection included adult and fetal hemoglobin and two functional candidate loci that were correlated with low hemoglobin concentration in Tibetans. Future detailed functional studies will now be required to examine the mechanistic underpinnings of physiological adaptation to high altitudes. Sequencing coding regions identified genetic changes that were likely involved in adaptation to hypoxia. Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18× per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNP’s association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.

Single-Cell Exome Sequencing Reveals Single-Nucleotide Mutation Characteristics of a Kidney Tumor

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and has very few mutations that are shared between different patients. To better understand the intratumoral genetics underlying mutations of ccRCC, we carried out single-cell exome sequencing on a ccRCC tumor and its adjacent kidney tissue. Our data indicate that this tumor was unlikely to have resulted from mutations in VHL and PBRM1. Quantitative population genetic analysis indicates that the tumor did not contain any significant clonal subpopulations and also showed that mutations that had different allele frequencies within the population also had different mutation spectrums. Analyses of these data allowed us to delineate a detailed intratumoral genetic landscape at a single-cell level. Our pilot study demonstrates that ccRCC may be more genetically complex than previously thought and provides information that can lead to new ways to investigate individual tumors, with the aim of developing more effective cellular targeted therapies.

TreeFam: a curated database of phylogenetic trees of animal gene families

TreeFam is a database of phylogenetic trees of gene families found in animals. It aims to develop a curated resource that presents the accurate evolutionary history of all animal gene families, as well as reliable ortholog and paralog assignments. Curated families are being added progressively, based on seed alignments and trees in a similar fashion to Pfam. Release 1.1 of TreeFam contains curated trees for 690 families and automatically generated trees for another 11 646 families. These represent over 128 000 genes from nine fully sequenced animal genomes and over 45 000 other animal proteins from UniProt; ∼40–85% of proteins encoded in the fully sequenced animal genomes are included in TreeFam. TreeFam is freely available at and .

Single-Cell Exome Sequencing and Monoclonal Evolution of a JAK2-Negative Myeloproliferative Neoplasm

Tumor heterogeneity presents a challenge for inferring clonal evolution and driver gene identification. Here, we describe a method for analyzing the cancer genome at a single-cell nucleotide level. To perform our analyses, we first devised and validated a high-throughput whole-genome single-cell sequencing method using two lymphoblastoid cell line single cells. We then carried out whole-exome single-cell sequencing of 90 cells from a JAK2-negative myeloproliferative neoplasm patient. The sequencing data from 58 cells passed our quality control criteria, and these data indicated that this neoplasm represented a monoclonal evolution. We further identified essential thrombocythemia (ET)-related candidate mutations such as SESN2 and NTRK1, which may be involved in neoplasm progression. This pilot study allowed the initial characterization of the disease-related genetic architecture at the single-cell nucleotide level. Further, we established a single-cell sequencing method that opens the way for detailed analyses of a variety of tumor types, including those with high genetic complex between patients.

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.

The DNA methylome of human peripheral blood mononuclear cells

Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.

Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants

Targeted capture combined with massively parallel exome sequencing is a promising approach to identify genetic variants implicated in human traits. We report exome sequencing of 200 individuals from Denmark with targeted capture of 18,654 coding genes and sequence coverage of each individual exome at an average depth of 12-fold. On average, about 95% of the target regions were covered by at least one read. We identified 121,870 SNPs in the sample population, including 53,081 coding SNPs (cSNPs). Using a statistical method for SNP calling and an estimation of allelic frequencies based on our population data, we derived the allele frequency spectrum of cSNPs with a minor allele frequency greater than 0.02. We identified a 1.8-fold excess of deleterious, non-syonomyous cSNPs over synonymous cSNPs in the low-frequency range (minor allele frequencies between 2% and 5%). This excess was more pronounced for X-linked SNPs, suggesting that deleterious substitutions are primarily recessive.

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.