Honglong Wu

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.

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.

Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1–50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1–23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.

An integrated epigenomic analysis for type 2 diabetes susceptibility loci in monozygotic twins

DNA methylation has a great potential for understanding the aetiology of common complex traits such as Type 2 diabetes (T2D). Here we perform genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-seq) in whole-blood-derived DNA from 27 monozygotic twin pairs and follow up results with replication and integrated omics analyses. We identify predominately hypermethylated T2D-related differentially methylated regions (DMRs) and replicate the top signals in 42 unrelated T2D cases and 221 controls. The strongest signal is in the promoter of the MALT1 gene, involved in insulin and glycaemic pathways, and related to taurocholate levels in blood. Integrating the DNA methylome findings with T2D GWAS meta-analysis results reveals a strong enrichment for DMRs in T2D-susceptibility loci. We also detect signals specific to T2D-discordant twins in the GPR61 and PRKCB genes. These replicated T2D associations reflect both likely causal and consequential pathways of the disease. The analysis indicates how an integrated genomics and epigenomics approach, utilizing an MZ twin design, can provide pathogenic insights as well as potential drug targets and biomarkers for T2D and other complex traits.

Hypermethylation in the ZBTB20 gene is associated with major depressive disorder

BackgroundAlthough genetic variation is believed to contribute to an individual’s susceptibility to major depressive disorder, genome-wide association studies have not yet identified associations that could explain the full etiology of the disease. Epigenetics is increasingly believed to play a major role in the development of common clinical phenotypes, including major depressive disorder.ResultsGenome-wide MeDIP-Sequencing was carried out on a total of 50 monozygotic twin pairs from the UK and Australia that are discordant for depression. We show that major depressive disorder is associated with significant hypermethylation within the coding region of ZBTB20, and is replicated in an independent cohort of 356 unrelated case-control individuals. The twins with major depressive disorder also show increased global variation in methylation in comparison with their unaffected co-twins. ZBTB20 plays an essential role in the specification of the Cornu Ammonis-1 field identity in the developing hippocampus, a region previously implicated in the development of major depressive disorder.ConclusionsOur results suggest that aberrant methylation profiles affecting the hippocampus are associated with major depressive disorder and show the potential of the epigenetic twin model in neuro-psychiatric disease.

Single-base-resolution methylomes of Populus trichocarpa reveal the association between DNA methylation and drought stress.

BackgroundDNA methylation is an important biological form of epigenetic modification, playing key roles in plant development and environmental responses.ResultsIn this study, we examined single-base resolution methylomes of Populus under control and drought stress conditions using high-throughput bisulfite sequencing for the first time. Our data showed methylation levels of methylated cytosines, upstream 2kp, downstream 2kb, and repeatitive sequences significantly increased after drought treatment in Populus. Interestingly, methylation in 100 bp upstream of the transcriptional start site (TSS) repressed gene expression, while methylations in 100-2000bp upstream of TSS and within the gene body were positively associated with gene expression. Integrated with the transcriptomic data, we found that all cis-splicing genes were non-methylated, suggesting that DNA methylation may not associate with cis-splicing. However, our results showed that 80% of trans-splicing genes were methylated. Moreover, we found 1156 transcription factors (TFs) with reduced methylation and expression levels and 690 TFs with increased methylation and expression levels after drought treatment. These TFs may play important roles in Populus drought stress responses through the changes of DNA methylation.ConclusionsThese findings may provide valuable new insight into our understanding of the interaction between gene expression and methylation of drought responses in Populus.

De novo assembly of a haplotype-resolved human genome

The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

Systematic assessment of reduced representation bisulfite sequencing to human blood samples: A promising method for large-sample-scale epigenomic studies

Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied reduced representation bisulfite sequencing (RRBS) to the human peripheral blood mononuclear cells (PBMC) from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility. Given a smaller data quantity, RRBS enjoys much better coverage depth than direct bisulfite sequencing and the concordance of DNA methylation levels between the two methods is high. It can be concluded that RRBS is a time and cost-effective sequencing method for unbiased DNA methylation profiling of CpG islands and promoter regions in a genome-wide scale and it is the method of choice to assay certain genomic regions for multiple samples in a rapid way.

Genome-Wide DNA Methylation Changes between the Superficial and Deep Backfat Tissues of the Pig

Adipose tissue is not only a storage organ involved in fuel metabolism, but also an endocrine organ involved in the regulation of insulin sensitivity, thermogenesis, immunity, and inflammation. There are anatomical, cellular, molecular and physiological differences among adipose tissues deposited in different body sites. However, current understanding of the intrinsic differences between the sub-compartments of the subcutaneous adipose tissue remains rudimentary. Here, we analyzed the genome-wide DNA methylation differences between the porcine superficial and deep backfat tissues using methylated DNA immunoprecipitation combined with high-throughput sequencing. We show that the genes with differentially methylated regions in their promoter are mainly involved in the processes of “lipid metabolism” and “regulation of immune-related cytokines”. Compared with the deep backfat tissue, the promoters of genes related to the ‘positive regulation of cytokine production’ were significantly hypermethylated in the superficial backfat tissue, which reflects the intrinsic functional and metabolic differences between the sub-compartments of the subcutaneous adipose tissue. This study provides epigenetic evidence for functionally relevant methylation differences between different layers of porcine backfat tissues.