The cost of whole-genome bisulfite sequencing (WGBS) remains a bottleneck for many studies and it is therefore imperative to extract as much information as possible from a given dataset. This is particularly important because even at the recommend 30X coverage for reference methylomes, up to 50% of high-resolution features such as differentially methylated positions (DMPs) cannot be called with current methods as determined by saturation analysis. To address this limitation, we have developed a tool that dynamically segments WGBS methylomes into blocks of comethylation (COMETs) from which lost information can be recovered in the form of differentially methylated COMETs (DMCs). Using this tool, we demonstrate recovery of ∼30% of the lost DMP information content as DMCs even at very low (5X) coverage. This constitutes twice the amount that can be recovered using an existing method based on differentially methylated regions (DMRs). In addition, we explored the relationship between COMETs and haplotypes in lymphoblastoid cell lines of African and European origin. Using best fit analysis, we show COMETs to be correlated in a population-specific manner, suggesting that this type of dynamic segmentation may be useful for integrated (epi)genome-wide association studies in the future.

Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity.

Exome sequencing has become a popular method to evaluate undirected mutagenesis experiments in mice. However, the most suitable mouse strain for the biological model may be relatively distant from the standard mouse reference genome. For pinpointing causative variants, a matching reference with gene annotations is essential, but not always readily available.

Modern horses represent heterogeneous populations specifically selected for appearance and performance. Genomic regions under high selective pressure show characteristic runs of homozygosity (ROH) which represent a low genetic diversity. This study aims at detecting the number and functional distribution of ROHs in different horse populations using next generation sequencing data.

Cactophilic Drosophila species provide a valuable model to study gene-environment interactions and ecological adaptation. Drosophila buzzatii and Drosophila mojavensis are two cactophilic species that belong to the repleta group, but have very different geographical distributions and primary host plants. To investigate the genomic basis of ecological adaptation, we sequenced the genome and developmental transcriptome of D. buzzatii and compared its gene content with that of D. mojavensis and two other noncactophilic Drosophila species in the same subgenus. The newly sequenced D. buzzatii genome (161.5 Mb) comprises 826 scaffolds (>3 kb) and contains 13,657 annotated protein-coding genes. Using RNA sequencing data of five life-stages we found expression of 15,026 genes, 80% protein-coding genes, and 20% noncoding RNA genes. In total, we detected 1,294 genes putatively under positive selection. Interestingly, among genes under positive selection in the D. mojavensis lineage, there is an excess of genes involved in metabolism of heterocyclic compounds that are abundant in Stenocereus cacti and toxic to nonresident Drosophila species. We found 117 orphan genes in the shared D. buzzatii-D. mojavensis lineage. In addition, gene duplication analysis identified lineage-specific expanded families with functional annotations associated with proteolysis, zinc ion binding, chitin binding, sensory perception, ethanol tolerance, immunity, physiology, and reproduction. In summary, we identified genetic signatures of adaptation in the shared D. buzzatii-D. mojavensis lineage, and in the two separate D. buzzatii and D. mojavensis lineages. Many of the novel lineage-specific genomic features are promising candidates for explaining the adaptation of these species to their distinct ecological niches.

T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.

Drosophila guanche is a member of the obscura group that originated in the Canary Islands archipelago upon its colonization by D. subobscura. It evolved into a new species in the laurisilva, a laurel forest present in wet regions that in the islands have only minor long-term weather fluctuations. Oceanic island endemic species such as D. guanche can become model species to investigate not only the relative role of drift and adaptation in speciation processes but also how population size affects nucleotide variation. Moreover, the previous identification of two satellite DNAs in D. guanche makes this species attractive for studying how centromeric DNA evolves. As a prerequisite for its establishment as a model species suitable to address all these questions, we generated a high-quality D. guanche genome sequence composed of 42 cytologically mapped scaffolds, which are assembled into six super-scaffolds (one per chromosome). The comparative analysis of the D. guanche proteome with that of twelve other Drosophila species identified 151 genes that were subject to adaptive evolution in the D. guanche lineage, with a subset of them being involved in flight and genome stability. For example, the Centromere Identifier (CID) protein, directly interacting with centromeric satellite DNA, shows signals of adaptation in this species. Both genomic analyses and FISH of the two satellites would support an ongoing replacement of centromeric satellite DNA in D. guanche.

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.

Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.

Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5' associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability.DOI: http://dx.doi.org/10.7554/eLife.02105.001.

After its domestication, rice cultivation expanded from tropical regions towards northern latitudes with temperate climate in a progressive process to overcome limiting photoperiod and temperature conditions. This process has originated a wide range of diversity that can be regarded as a valuable resource for crop improvement. In general, current rice breeding programs have to deal with a lack of both germplasm accessions specifically adapted to local agro-environmental conditions and adapted donors carrying desired agronomical traits. Comprehensive maps of genome variability and population structure would facilitate genome-wide association studies of complex traits, functional gene investigations and the selection of appropriate donors for breeding purposes.

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.

Six extant species of non-human great apes are currently recognized: Sumatran and Bornean orangutans, eastern and western gorillas, and chimpanzees and bonobos [1]. However, large gaps remain in our knowledge of fine-scale variation in hominoid morphology, behavior, and genetics, and aspects of great ape taxonomy remain in flux. This is particularly true for orangutans (genus: Pongo), the only Asian great apes and phylogenetically our most distant relatives among extant hominids [1]. Designation of Bornean and Sumatran orangutans, P. pygmaeus (Linnaeus 1760) and P. abelii (Lesson 1827), as distinct species occurred in 2001 [1, 2]. Here, we show that an isolated population from Batang Toru, at the southernmost range limit of extant Sumatran orangutans south of Lake Toba, is distinct from other northern Sumatran and Bornean populations. By comparing cranio-mandibular and dental characters of an orangutan killed in a human-animal conflict to those of 33 adult male orangutans of a similar developmental stage, we found consistent differences between the Batang Toru individual and other extant Ponginae. Our analyses of 37 orangutan genomes provided a second line of evidence. Model-based approaches revealed that the deepest split in the evolutionary history of extant orangutans occurred ∼3.38 mya between the Batang Toru population and those to the north of Lake Toba, whereas both currently recognized species separated much later, about 674 kya. Our combined analyses support a new classification of orangutans into three extant species. The new species, Pongo tapanuliensis, encompasses the Batang Toru population, of which fewer than 800 individuals survive. VIDEO ABSTRACT.

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

Bringing together cancer genomes from different projects increases power and allows the investigation of pan-cancer, molecular mechanisms. However, working with whole genomes sequenced over several years in different sequencing centres requires a framework to compare the quality of these sequences. We used the Pan-Cancer Analysis of Whole Genomes cohort as a test case to construct such a framework. This cohort contains whole cancer genomes of 2832 donors from 18 sequencing centres. We developed a non-redundant set of five quality control (QC) measurements to establish a star rating system. These QC measures reflect known differences in sequencing protocol and provide a guide to downstream analyses and allow for exclusion of samples of poor quality. We have found that this is an effective framework of quality measures. The implementation of the framework is available at: https://dockstore.org/containers/quay.io/jwerner_dkfz/pancanqc:1.2.2 .

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 1.5 million fatalities since it first emerged in late 2019. As we write, infection rates are currently at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary viral target is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predicts that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results and finds additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and Endangered aye-aye ( Daubentonia madagascariensis ), as well as those in the genera Avahi and Propithecus , may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.