Vam6/Vps39/TRAP1-domain proteins influence vacuolar morphology, iron acquisition and virulence in Cryptococcus neoformans.

The pathogenic fungus Cryptococcus neoformans must overcome iron limitation to cause disease in mammalian hosts. Previously, we reported a screen for insertion mutants with poor growth on haem as the sole iron source. In this study, we characterised one such mutant and found that the defective gene encoded a Vam6/Vps39/TRAP1 domain-containing protein required for robust growth on haem, an important iron source in host tissue. We designated this protein Vps3 based on reciprocal best matches with the corresponding protein in Saccharomyces cerevisiae. C. neoformans encodes a second Vam6/Vps39/TRAP1 domain-containing protein designated Vam6/Vlp1, and we found that this protein is also required for robust growth on haem as well as on inorganic iron sources. This protein is predicted to be a component of the homotypic fusion and vacuole protein sorting complex involved in endocytosis. Further characterisation of the vam6Δ and vps3Δ mutants revealed perturbed trafficking of iron acquisition functions (e.g., the high affinity iron permease Cft1) and impaired processing of the transcription factor Rim101, a regulator of haem and iron acquisition. The vps3Δ and vam6Δ mutants also had pleiotropic phenotypes including loss of virulence in a mouse model of cryptococcosis, reduced virulence factor elaboration and increased susceptibility to stress, indicating pleiotropic roles for Vps3 and Vam6 beyond haem use in C. neoformans. TAKE AWAYS: Two Vam6/Vps39/TRAP1-domain proteins, Vps3 and Vam6, support the growth of Cryptococcus neoformans on haem. Loss of Vps3 and Vam6 influences the trafficking and expression of iron uptake proteins. Loss of Vps3 or Vam6 eliminates the ability of C. neoformans to cause disease in a mouse model of cryptococcosis.

Evidence for opposing selective forces operating on human-specific duplicated TCAF genes in Neanderthals and humans.

TRP channel-associated factor 1/2 (TCAF1/TCAF2) proteins antagonistically regulate the cold-sensor protein TRPM8 in multiple human tissues. Understanding their significance has been complicated given the locus spans a gap-ridden region with complex segmental duplications in GRCh38. Using long-read sequencing, we sequence-resolve the locus, annotate full-length TCAF models in primate genomes, and show substantial human-specific TCAF copy number variation. We identify two human super haplogroups, H4 and H5, and establish that TCAF duplications originated ~1.7 million years ago but diversified only in Homo sapiens by recurrent structural mutations. Conversely, in all archaic-hominin samples the fixation for a specific H4 haplotype without duplication is likely due to positive selection. Here, our results of TCAF copy number expansion, selection signals in hominins, and differential TCAF2 expression between haplogroups and high TCAF2 and TRPM8 expression in liver and prostate in modern-day humans imply TCAF diversification among hominins potentially in response to cold or dietary adaptations.

A high-quality bonobo genome refines the analysis of hominid evolution.

The divergence of chimpanzee and bonobo provides one of the few examples of recent hominid speciation1,2. Here we describe a fully annotated, high-quality bonobo genome assembly, which was constructed without guidance from reference genomes by applying a multiplatform genomics approach. We generate a bonobo genome assembly in which more than 98% of genes are completely annotated and 99% of the gaps are closed, including the resolution of about half of the segmental duplications and almost all of the full-length mobile elements. We compare the bonobo genome to those of other great apes1,3-5 and identify more than 5,569 fixed structural variants that specifically distinguish the bonobo and chimpanzee lineages. We focus on genes that have been lost, changed in structure or expanded in the last few million years of bonobo evolution. We produce a high-resolution map of incomplete lineage sorting and estimate that around 5.1% of the human genome is genetically closer to chimpanzee or bonobo and that more than 36.5% of the genome shows incomplete lineage sorting if we consider a deeper phylogeny including gorilla and orangutan. We also show that 26% of the segments of incomplete lineage sorting between human and chimpanzee or human and bonobo are non-randomly distributed and that genes within these clustered segments show significant excess of amino acid replacement compared to the rest of the genome.

The structure, function and evolution of a complete human chromosome 8.

The complete assembly of each human chromosome is essential for understanding human biology and evolution1,2. Here we use complementary long-read sequencing technologies to complete the linear assembly of human chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08-Mb centromeric α-satellite array, a 644-kb copy number polymorphism in the ß-defensin gene cluster that is important for disease risk, and an 863-kb variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73-kb hypomethylated region of diverse higher-order α-satellites enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. In addition, we confirm the overall organization and methylation pattern of the centromere in a diploid human genome. Using a dual long-read sequencing approach, we complete high-quality draft assemblies of the orthologous centromere from chromosome 8 in chimpanzee, orangutan and macaque to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved in the great ape ancestor with a layered symmetry, in which more ancient higher-order repeats locate peripherally to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated by more than 2.2-fold compared to the unique portions of the genome, and this acceleration extends into the flanking sequence.

Fully phased human genome assembly without parental data using single-cell strand sequencing and long reads.

Human genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.

An evolutionary driver of interspersed segmental duplications in primates.

BACKGROUND: The complex interspersed pattern of segmental duplications in humans is responsible for rearrangements associated with neurodevelopmental disease, including the emergence of novel genes important in human brain evolution. We investigate the evolution of LCR16a, a putative driver of this phenomenon that encodes one of the most rapidly evolving human-ape gene families, nuclear pore interacting protein (NPIP). RESULTS: Comparative analysis shows that LCR16a has independently expanded in five primate lineages over the last 35 million years of primate evolution. The expansions are associated with independent lineage-specific segmental duplications flanking LCR16a leading to the emergence of large interspersed duplication blocks at non-orthologous chromosomal locations in each primate lineage. The intron-exon structure of the NPIP gene family has changed dramatically throughout primate evolution with different branches showing characteristic gene models yet maintaining an open reading frame. In the African ape lineage, we detect signatures of positive selection that occurred after a transition to more ubiquitous expression among great ape tissues when compared to Old World and New World monkeys. Mouse transgenic experiments from baboon and human genomic loci confirm these expression differences and suggest that the broader ape expression pattern arose due to mutational changes that emerged in cis. CONCLUSIONS: LCR16a promotes serial interspersed duplications and creates hotspots of genomic instability that appear to be an ancient property of primate genomes. Dramatic changes to NPIP gene structure and altered tissue expression preceded major bouts of positive selection in the African ape lineage, suggestive of a gene undergoing strong adaptive evolution.

Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.

Recurrent inversion toggling and great ape genome evolution.

Inversions play an important role in disease and evolution but are difficult to characterize because their breakpoints map to large repeats. We increased by sixfold the number (n = 1,069) of previously reported great ape inversions by using single-cell DNA template strand and long-read sequencing. We find that the X chromosome is most enriched (2.5-fold) for inversions, on the basis of its size and duplication content. There is an excess of differentially expressed primate genes near the breakpoints of large (>100 kilobases (kb)) inversions but not smaller events. We show that when great ape lineage-specific duplications emerge, they preferentially (approximately 75%) occur in an inverted orientation compared to that at their ancestral locus. We construct megabase-pair scale haplotypes for individual chromosomes and identify 23 genomic regions that have recurrently toggled between a direct and an inverted state over 15 million years. The direct orientation is most frequently the derived state for human polymorphisms that predispose to recurrent copy number variants associated with neurodevelopmental disease.

Evolutionary Dynamics of the POTE Gene Family in Human and Nonhuman Primates.

POTE (prostate, ovary, testis, and placenta expressed) genes belong to a primate-specific gene family expressed in prostate, ovary, and testis as well as in several cancers including breast, prostate, and lung cancers. Due to their tumor-specific expression, POTEs are potential oncogenes, therapeutic targets, and biomarkers for these malignancies. This gene family maps within human and primate segmental duplications with a copy number ranging from two to 14 in different species. Due to the high sequence identity among the gene copies, specific efforts are needed to assemble these loci in order to correctly define the organization and evolution of the gene family. Using single-molecule, real-time (SMRT) sequencing, in silico analyses, and molecular cytogenetics, we characterized the structure, copy number, and chromosomal distribution of the POTE genes, as well as their expression in normal and disease tissues, and provided a comparative analysis of the POTE organization and gene structure in primate genomes. We were able, for the first time, to de novo sequence and assemble a POTE tandem duplication in marmoset that is misassembled and collapsed in the reference genome, thus revealing the presence of a second POTE copy. Taken together, our findings provide comprehensive insights into the evolutionary dynamics of the primate-specific POTE gene family, involving gene duplications, deletions, and long interspersed nuclear element (LINE) transpositions to explain the actual repertoire of these genes in human and primate genomes.

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes.

Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.

Characterizing the Major Structural Variant Alleles of the Human Genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.

Long-read sequence and assembly of segmental duplications.

We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.

Microbial and geochemical controls on waste rock weathering and drainage quality.

Bacteria can adversely affect the quality of drainage released from mine waste by catalyzing the oxidation of sulfide minerals and thereby accelerating the release of acidity and metals. However, the microbiological and geochemical controls on drainage quality from unsaturated and geochemically heterogeneous waste rock remain poorly understood. Here, we identified coexisting neutrophilic and acidophilic bacteria in different types of waste rock, indicating that robust endemic consortia are sustained within pore-scale microenvironments. Subsequently, natural weathering was simulated in laboratory column experiments with waste rock that contained either in-situ microbial consortia or suppressed populations with up to 1000 times smaller abundance and reduced phenotypic diversity after heating and drying. Drainage from waste rock with in-situ populations was up to two pH units lower and contained up to 16 times more sulfate and heavy metals compared to drainage from waste rock bearing treated populations, indicating significantly higher sulfide-oxidation rates. The drainage chemistry was further affected by sorption and formation of secondary-mineral phases (e.g., gypsum and hydroxy-carbonates). This study provides direct evidence for the existence of diverse microbial communities in waste rock and their important catalytic role on weathering rates, and illustrates the mutual controls of microbiology and geochemistry on waste-rock drainage quality.

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ∼25-35 Mya and CFHR1 and CFHR3 ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.