Ian T Fiddes

Improved data analysis for the MinION nanopore sequencer

Speed, single-base sensitivity and long read lengths make nanopores a promising technology for high-throughput sequencing. We evaluated and optimized the performance of the MinION nanopore sequencer using M13 genomic DNA and used expectation maximization to obtain robust maximum-likelihood estimates for insertion, deletion and substitution error rates (4.9%, 7.8% and 5.1%, respectively). Over 99% of high-quality 2D MinION reads mapped to the reference at a mean identity of 85%. We present a single-nucleotide-variant detection tool that uses maximum-likelihood parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer-testis gene family (CT47) within an unresolved region of human chromosome Xq24.

Long-read sequence assembly of the gorilla genome

Improving on the gorilla genome Access to complete, high-quality genomes of nonhuman primates will also help us understand human biology. Gordon et al. used long-read sequencing technology to improve genome data on our close relative the gorilla. Sequencing from a single individual decreased assembly fragmentation and recovered previously missed genes and noncoding loci. Mapping short-read sequences from additional gorillas helped reconstruct a “pan” gorilla sequence documenting genetic variation. Comparison with human genomes revealed species-specific differences ranging in size from one to thousands of bases in length, including some that are likely to affect gene regulation. Science, this issue p. 10.1126/science.aae0344 A new approach to looking at the gorilla genome improves estimates of the differences between humans and gorillas. INTRODUCTION The accurate sequence and assembly of genomes is critical to our understanding of evolution and genetic variation. Despite advances in short-read sequencing technology that have decreased cost and increased throughput, whole-genome assembly of mammalian genomes remains problematic because of the presence of repetitive DNA. RATIONALE The goal of this study was to sequence and assemble the genome of the western lowland gorilla by using primarily single-molecule, real-time (SMRT) sequencing technology and a novel assembly algorithm that takes advantage of long (>10 kbp) sequence reads. We specifically compare the properties of this assembly to gorilla genome assemblies that were generated by using more routine short sequence read approaches in order to determine the value and biological impact of a long-read genome assembly. RESULTS We generated 74.8-fold SMRT whole-genome shotgun sequence from peripheral blood DNA isolated from a western lowland gorilla (Gorilla gorilla gorilla) named Susie. We applied a string graph assembly algorithm, Falcon, and consensus algorithm, Quiver, to generate a 3.1-Gbp assembly with a contig N50 of 9.6 Mbp. Short-read sequence data from an additional six gorilla genomes was mapped so as to reduce indel errors and improve the accuracy of the final assembly. We estimate that 98.9% of the gorilla euchromatin has been assembled into 1854 sequence contigs. The assembly represents an improvement in contiguity: >800-fold with respect to the published gorilla genome assembly and >180-fold with respect to a more recently released upgrade of the gorilla assembly. Most of the sequence gaps are now closed, considerably increasing the yield of complete gene models. We estimate that 87% of the missing exons and 94% of the incomplete genes are recovered. We find that the sequence of most full-length common repeats is resolved, with the most significant gains occurring for the longest and most G+C–rich retrotransposons. Although complex regions such as the major histocompatibility locus are accurately sequenced and assembled, both heterochromatin and large, high-identity segmental duplications are not because read lengths are insufficiently long to traverse these repetitive structures. The long-read assembly produces a much finer map of structural variation down to 50 bp in length, facilitating the discovery of thousands of lineage-specific structural variant differences that have occurred since divergence from the human and chimpanzee lineages. This includes the disruption of specific genes and loss of predicted regulatory regions between the two species. We show that use of the new gorilla genome assembly changes estimates of divergence and diversity, resulting in subtle but substantial effects on previous population genetic inferences, such as the timing of species bottlenecks and changes in the effective population size over the course of evolution. CONCLUSION The genome assembly that results from using the long-read data provides a more complete picture of gene content, structural variation, and repeat biology, improving population genetic and evolutionary inferences. Long-read sequencing technology now makes it practical for individual laboratories to generate high-quality reference genomes for complex mammalian genomes. Long-read sequence assembly of the gorilla genome. (A) Susie, a female Western lowland gorilla, was used as the reference sample for full-genome sequencing and assembly [photograph courtesy of Max Block]. (B and C) A treemaps representing the differences in fragmentation of the long-read and short-read gorilla genome assemblies. The rectangles are the largest contigs that cumulatively make up 300 Mbp (~10%) of the assembly. Accurate sequence and assembly of genomes is a critical first step for studies of genetic variation. We generated a high-quality assembly of the gorilla genome using single-molecule, real-time sequence technology and a string graph de novo assembly algorithm. The new assembly improves contiguity by two to three orders of magnitude with respect to previously released assemblies, recovering 87% of missing reference exons and incomplete gene models. Although regions of large, high-identity segmental duplications remain largely unresolved, this comprehensive assembly provides new biological insight into genetic diversity, structural variation, gene loss, and representation of repeat structures within the gorilla genome. The approach provides a path forward for the routine assembly of mammalian genomes at a level approaching that of the current quality of the human genome.

Nanopore sequencing and assembly of a human genome with ultra-long reads

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

The UCSC Genome Browser database: 2018 update.

Abstract The UCSC Genome Browser (https://genome.ucsc.edu) provides a web interface for exploring annotated genome assemblies. The assemblies and annotation tracks are updated on an ongoing basis—12 assemblies and more than 28 tracks were added in the past year. Two recent additions are a display of CRISPR/Cas9 guide sequences and an interactive navigator for gene interactions. Other upgrades from the past year include a command-line version of the Variant Annotation Integrator, support for Human Genome Variation Society variant nomenclature input and output, and a revised highlighting tool that now supports multiple simultaneous regions and colors.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

High-resolution comparative analysis of great ape genomes

A spotlight on great ape genomes Most nonhuman primate genomes generated to date have been “humanized” owing to their many gaps and the reliance on guidance by the reference human genome. To remove this humanizing effect, Kronenberg et al. generated and assembled long-read genomes of a chimpanzee, an orangutan, and two humans and compared them with a previously generated gorilla genome. This analysis recognized genomic structural variation specific to humans and particular ape lineages. Comparisons between human and chimpanzee cerebral organoids showed down-regulation of the expression of specific genes in humans, relative to chimpanzees, related to noncoding variation identified in this analysis. Science, this issue p. eaar6343 Analysis of long-read great ape and human genomes identifies human-specific changes affecting brain gene expression. INTRODUCTION Understanding the genetic differences that make us human is a long-standing endeavor that requires the comprehensive discovery and comparison of all forms of genetic variation within great ape lineages. RATIONALE The varied quality and completeness of ape genomes have limited comparative genetic analyses. To eliminate this contiguity and quality disparity, we generated human and nonhuman ape genome assemblies without the guidance of the human reference genome. These new genome assemblies enable both coarse and fine-scale comparative genomic studies. RESULTS We sequenced and assembled two human, one chimpanzee, and one orangutan genome using high-coverage (>65x) single-molecule, real-time (SMRT) long-read sequencing technology. We also sequenced more than 500,000 full-length complementary DNA samples from induced pluripotent stem cells to construct de novo gene models, increasing our knowledge of transcript diversity in each ape lineage. The new nonhuman ape genome assemblies improve gene annotation and genomic contiguity (by 30- to 500-fold), resulting in the identification of larger synteny blocks (by 22- to 74-fold) when compared to earlier assemblies. Including the latest gorilla genome, we now estimate that 83% of the ape genomes can be compared in a multiple sequence alignment. We observe a modest increase in single-nucleotide variant divergence compared to previous genome analyses and estimate that 36% of human autosomal DNA is subject to incomplete lineage sorting. We fully resolve most common repeat differences, including full-length retrotransposons such as the African ape-specific endogenous retroviral element PtERV1. We show that the spread of this element independently in the gorilla and chimpanzee lineage likely resulted from a founder element that failed to segregate to the human lineage because of incomplete lineage sorting. The improved sequence contiguity allowed a more systematic discovery of structural variation (>50 base pairs in length) (see the figure). We detected 614,186 ape deletions, insertions, and inversions, assigning each to specific ape lineages. Unbiased genome scaffolding (optical maps, bacterial artificial chromosome sequencing, and fluorescence in situ hybridization) led to the discovery of large, unknown complex inversions in gene-rich regions. Of the 17,789 fixed human-specific insertions and deletions, we focus on those of potential functional effect. We identify 90 that are predicted to disrupt genes and an additional 643 that likely affect regulatory regions, more than doubling the number of human-specific deletions that remove regulatory sequence in the human lineage. We investigate the association of structural variation with changes in human-chimpanzee brain gene expression using cerebral organoids as a proxy for expression differences. Genes associated with fixed structural variants (SVs) show a pattern of down-regulation in human radial glial neural progenitors, whereas human-specific duplications are associated with up-regulated genes in human radial glial and excitatory neurons (see the figure). CONCLUSION The improved ape genome assemblies provide the most comprehensive view to date of intermediate-size structural variation and highlight several dozen genes associated with structural variation and brain-expression differences between humans and chimpanzees. These new references will provide a stepping stone for the completion of great ape genomes at a quality commensurate with the human reference genome and, ultimately, an understanding of the genetic differences that make us human. SMRT assemblies and SV analyses. (Top) Contiguity of the de novo assemblies. (Bottom, left to right) For each ape, SVdetection was done against the human reference genome as represented by a dot plot of an inversion). Human-specific SVs, identified by comparing ape SVs and population genotyping (0/0, homozygous reference),were compared to single-cell gene expression differences [range: low (dark blue) to high (dark red)] in primary and organoid tissues. Each heatmap row is a gene that intersects an insertion or deletion (green), duplication (cyan), or inversion (light green). 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.

Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like−/− mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

Evaluating recovery potential of the northern white rhinoceros from cryopreserved somatic cells

The critically endangered northern white rhinoceros is believed to be extinct in the wild, with the recent death of the last male leaving only two remaining individuals in captivity. Its extinction would appear inevitable, but the development of advanced cell and reproductive technologies such as cloning by nuclear transfer and the artificial production of gametes via stem cells differentiation offer a second chance for its survival. In this work, we analyzed genome-wide levels of genetic diversity, inbreeding, population history, and demography of the white rhinoceros sequenced from cryopreserved somatic cells, with the goal of informing how genetically valuable individuals could be used in future efforts toward the genetic rescue of the northern white rhinoceros. We present the first sequenced genomes of the northern white rhinoceros, which show relatively high levels of heterozygosity and an average genetic divergence of 0.1% compared with the southern subspecies. The two white rhinoceros subspecies appear to be closely related, with low genetic admixture and a divergent time <80,000 yr ago. Inbreeding, as measured by runs of homozygosity, appears slightly higher in the southern than the northern white rhinoceros. This work demonstrates the value of the northern white rhinoceros cryopreserved genetic material as a potential gene pool for saving this subspecies from extinction.

A 3-way hybrid approach to generate a new high-quality chimpanzee reference genome (Pan_tro_3.0)

Abstract The chimpanzee is arguably the most important species for the study of human origins. A key resource for these studies is a high-quality reference genome assembly; however, as with most mammalian genomes, the current iteration of the chimpanzee reference genome assembly is highly fragmented. In the current iteration of the chimpanzee reference genome assembly (Pan_tro_2.1.4), the sequence is scattered across more then 183 000 contigs, incorporating more than 159 000 gaps, with a genome-wide contig N50 of 51 Kbp. In this work, we produce an extensive and diverse array of sequencing datasets to rapidly assemble a new chimpanzee reference that surpasses previous iterations in bases represented and organized in large scaffolds. To this end, we show substantial improvements over the current release of the chimpanzee genome (Pan_tro_2.1.4) by several metrics, such as increased contiguity by >750% and 300% on contigs and scaffolds, respectively, and closure of 77% of gaps in the Pan_tro_2.1.4 assembly gaps spanning >850 Kbp of the novel coding sequence based on RNASeq data. We further report more than 2700 genes that had putatively erroneous frame-shift predictions to human in Pan_tro_2.1.4 and show a substantial increase in the annotation of repetitive elements. We apply a simple 3-way hybrid approach to considerably improve the reference genome assembly for the chimpanzee, providing a valuable resource for the study of human origins. Furthermore, we produce extensive sequencing datasets that are all derived from the same cell line, generating a broad non-human benchmark dataset.