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The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20–30 cancer genes 1 – 8 . Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease 9 – 13 than in normal liver 13 – 16 , which enables positive selection to shape the genomic landscape 9 – 13 . Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1 , the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1 S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB , which regulates lipid droplet metabolism in hepatocytes 17 – 19 , and GPAM , which produces storage triacylglycerol from free fatty acids 20 , 21 , also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease. Whole-genome sequencing analysis of somatic mutations in liver samples from patients with chronic liver disease identifies driver mutations in metabolism-related genes such as FOXO1 , and shows that these variants frequently exhibit convergent evolution.

Background Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90–95%. Results We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5′ and 3′ untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts. Conclusions The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5′ and 3′ ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.

Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol “DAFT-seq” to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.

The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known 'fragile sites'. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes-including a large proportion of vars-showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen.

Germ cell tumours (GCTs) are a collection of benign and malignant neoplasms derived from primordial germ cells. They are uniquely able to recapitulate embryonic and extraembryonic tissues, which carries prognostic and therapeutic significance. The developmental pathways underpinning GCT initiation and histogenesis are incompletely understood. Here, we study the relationship of histogenesis and clonal diversification in GCTs by analysing the genomes and transcriptomes of 547 microdissected histological units. We find no correlation between genomic and histological heterogeneity. However, we identify unifying features including the retention of fetal developmental transcripts across tissues, expression changes on chromosome 12p, and a conserved somatic evolutionary sequence of whole genome duplication followed by clonal diversification. While this pattern is preserved across all GCTs, the developmental timing of the duplication varies between prepubertal and postpubertal cases. In addition, tumours of younger children exhibit distinct substitution signatures which may lend themselves as potential biomarkers for risk stratification. Our findings portray the extensive diversification of GCT tissues and genetic subclones as randomly distributed, while identifying overarching transcriptional and genomic features. The molecular characterisation of germ cell tumours (GCT) is necessary to understand their development and histological diversification. Here, the authors use whole-genome and transcriptome sequencing of GCTs across distinct histologies to reveal their somatic evolution and clonal diversification, as well as identify several putative biomarkers for treatment stratification.

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

Background Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum , generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes. Results Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2 , proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates. Conclusions Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.

Messenger RNA is modified by the addition of a 5' methylated cap structure, which protects the transcript and recruits protein complexes that mediate RNA processing and/or the initiation of translation. Two genes encoding mRNA cap methyltransferases have been identified in T. brucei: TbCMT1 and TbCGM1. Here we analysed the impact of TbCMT1 gene deletion on bloodstream form T. brucei cells. TbCMT1 was dispensable for parasite proliferation in in vitro culture. However, significantly decreased parasitemia was observed in mice inoculated with TbCMT1 null and conditional null cell lines. Using RNA-Seq, we observed that several cysteine peptidase mRNAs were downregulated in TbCMT1 null cells lines. The cysteine peptidase Cathepsin-L was also shown to be reduced at the protein level in TbCMT1 null cell lines. Our data suggest that TbCMT1 is not essential to bloodstream form T. brucei growth in vitro or in vivo but that it contributes significantly to parasite virulence in vivo.

This month's Genome Watch highlights a recent study that used high-throughput sequencing to identify the mRNAs that are actively translated during the life cycle of a protozoan parasite. This month's Genome Watch highlights a recent study that used high-throughput sequencing to identify the mRNAs that are actively translated during the life cycle of a protozoan parasite.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial–mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo’s architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial–mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo. Sozen et al. devise an approach to combine embryonic stem cells, trophoblast stem cells and extra-embryonic endoderm stem cells into self-assembling embryo-like structures, which recapitulate key hallmarks of gastrulation in vitro.