Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Identification of essential genes in C. elegans through whole genome sequencing of legacy mutant collections

It has been estimated that 15-30% of the ~20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with sperm-specific functions. In total, 59 essential genes were identified on chromosomes III, IV, and V, and 20 of these genes (42 alleles) were selected for further bioinformatic and functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown sperm-expressed genes required for fertilization and embryonic development. The result of this study is a catalogue of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.Medicine, Faculty ofGraduat

A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen’s design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10−4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations

CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans

The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans. We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events

Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT)

<div><p>Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using <i>C</i>. <i>elegans</i> as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the <i>trans</i>-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.</p></div

Confirmation of <i>bgnt-1</i>.<i>1</i> as a novel dye-filling gene.

<p>(A) Wild-type <i>bgnt-1</i>.<i>1</i> rescues dye-filling defects in <i>bgnt-1</i>.<i>1 (gk361915)</i> mutants. Transformation of VC20628 <i>bgnt-1</i>.<i>1 (gk361915)</i> with a fosmid containing wild-type <i>bgnt-1</i>.<i>1</i> (WRM065bB05) completely rescues the amphid, and partially rescues the phasmid dye-filling defects of VC20628 <i>bgnt-1</i>.<i>1 (gk361915)</i> mutants, as assessed by fluorescence microscopy. (B) Quantitation of amphid and phasmid dye-filling in the mutant strain VC20628 in the presence or absence of a fosmid rescue construct (p < 0.05, Fisher’s exact test). Error bars represent 95% confidence intervals (Pearson Clopper method).</p

Dye-filling (ciliated sensory neuron development/function) screening methodology and results.

<p>(A, B) Input to the screen was 480 whole genome-sequenced multi-mutant strains from the Million Mutation Project [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006235#pgen.1006235.ref010" target="_blank">10</a>]. Mixed-stage <i>C</i>. <i>elegans</i> cultures were incubated with DiI for 30 minutes, washed in buffer and then examined by fluorescence microscopy for their ability to uptake the dye into head (amphid) and tail (phasmid) sensory neurons. (C) Dye-filling phenotypes of each of the 480 MMP strains which were assayed. The proportion of worms exhibiting dye-filling in strains represented by dark grey diamonds were not statistically separable from the proportion of wild-type worms exhibiting dye-filling as assessed by a Fisher’s exact test with p-values adjusted for a 5% false discovery rate (Benjamini–Hochberg procedure) to control for multiple testing. Blue and red diamonds represent strains with mainly or exclusively amphid or phasmid dye-filling defects, respectively, while purple diamonds show strains with defects in both amphid and phasmid sensory neurons. Two highlighted strains, VC20615 and VC20628, contain mutations which alter conserved amino acid residues in the protein encoded by <i>C</i>. <i>elegans bgnt-1</i>.<i>1</i>, a gene identified by SKAT to be associated with both amphid and phasmid dye-filling defects.</p

Genes with genome-wide significance for phasmid ciliated neuron dye-filling phenotypes, ordered by FDR adjusted p-value.

<p>Genes with genome-wide significance for phasmid ciliated neuron dye-filling phenotypes, ordered by FDR adjusted p-value.</p

CRISPR-Cas9 knockout of <i>bgnt-1</i>.<i>1</i> causes dye-filling defects.

<p>(A) three <i>bgnt-1</i>.<i>1</i> knockout alleles, independently generated <i>via</i> CRISPR-Cas9, exhibit amphid and phasmid (B) dye-fill defects compared to wild-type worms (p < 0.0001, Fisher’s exact test, p-values adjusted for a 5% false discovery rate using the Benjamini–Hochberg procedure). The magnitude of the dye-filling defects in the CRISPR-Cas9 knockout alleles is similar to that observed in the MMP <i>bgnt-1</i>.<i>1 gk210889</i> missense mutation, which results in G205E amino acid change. Error bars represent 95% confidence intervals (Pearson Clopper method). (C) Representative images of dye-filling in wild-type (N2), three CRISPR-Cas9-generated <i>bgnt-1</i>.<i>1</i> knockout alleles and the MMP <i>bgnt-1</i>.<i>1 gk210889</i> missense mutation allele.</p