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April 21, 2020  |  

Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes. Copyright © 2019 Elsevier Inc. All rights reserved.


April 21, 2020  |  

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.


April 21, 2020  |  

Urotensin-related gene transcripts mark developmental emergence of the male forebrain vocal control system in songbirds.

Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication.


April 21, 2020  |  

Chromosome-level assembly of the water buffalo genome surpasses human and goat genomes in sequence contiguity.

Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5?kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly more than a thousand-fold and contains only 383 gaps. It surpasses the human and goat references in sequence contiguity and facilitates the annotation of hard to assemble gene clusters such as the major histocompatibility complex (MHC).


April 21, 2020  |  

The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes.

Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock.Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123?Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7?Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons.This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines.


October 23, 2019  |  

The genome of common long-arm octopus Octopus minor.

The common long-arm octopus (Octopus minor) is found in mudflats of subtidal zones and faces numerous environmental challenges. The ability to adapt its morphology and behavioral repertoire to diverse environmental conditions makes the species a promising model for understanding genomic adaptation and evolution in cephalopods.The final genome assembly of O. minor is 5.09 Gb, with a contig N50 size of 197 kb and longest size of 3.027 Mb, from a total of 419 Gb raw reads generated using the Pacific Biosciences RS II platform. We identified 30,010 genes; 44.43% of the genome is composed of repeat elements. The genome-wide phylogenetic tree indicated the divergence time between O. minor and Octopus bimaculoides was estimated to be 43 million years ago based on single-copy orthologous genes. In total, 178 gene families are expanded in O. minor in the 14 bilaterian species.We found that the O. minor genome was larger than that of closely related O. bimaculoides, and this difference could be explained by enlarged introns and recently diversified transposable elements. The high-quality O. minor genome assembly provides a valuable resource for understanding octopus genome evolution and the molecular basis of adaptations to mudflats.


September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.


September 22, 2019  |  

De novo assembly and phasing of dikaryotic genomes from two isolates of Puccinia coronata f. sp. avenae, the causal agent of oat crown rust.

Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae, is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenaeIMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae, which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae, resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae. Copyright © 2018 Miller et al.


September 22, 2019  |  

The complete chloroplast genome of Chrysanthemum boreale (Asteraceae)

Chrysanthemum boreale is a perennial plant in the Asteraceae family that is native to eastern Asia and has both ornamental and herbal uses. Here, we determined the complete chloroplast genome sequence for C. boreale using long-read sequencing. The chloroplast genome was 151,012?bp and consisted of a large single copy (LSC) region (82,817?bp), a small single copy (SSC) region (18,281?bp) and two inverted repeats (IRs) (24,957?bp). It was predicted to contain 131 genes, including 87 protein-coding genes, eight rRNAs and 46 tRNAs. Phylogenetic analysis of chloroplast genomes clustered C. boreale with other Chrysanthemum and Asteraceae species.


September 22, 2019  |  

Assembling the genome of the African wild rice Oryza longistaminata by exploiting synteny in closely related Oryza species.

The African wild rice species Oryza longistaminata has several beneficial traits compared to cultivated rice species, such as resistance to biotic stresses, clonal propagation via rhizomes, and increased biomass production. To facilitate breeding efforts and functional genomics studies, we de-novo assembled a high-quality, haploid-phased genome. Here, we present our assembly, with a total length of 351?Mb, of which 92.2% was anchored onto 12 chromosomes. We detected 34,389 genes and 38.1% of the genome consisted of repetitive content. We validated our assembly by a comparative linkage analysis and by examining well-characterized gene families. This genome assembly will be a useful resource to exploit beneficial alleles found in O. longistaminata. Our results also show that it is possible to generate a high-quality, functionally complete rice genome assembly from moderate SMRT read coverage by exploiting synteny in a closely related Oryza species.


September 22, 2019  |  

Genomic analysis of Picochlorum species reveals how microalgae may adapt to variable environments.

Understanding how microalgae adapt to rapidly changing environments is not only important to science but can help clarify the potential impact of climate change on the biology of primary producers. We sequenced and analyzed the nuclear genome of multiple Picochlorum isolates (Chlorophyta) to elucidate strategies of environmental adaptation. It was previously found that coordinated gene regulation is involved in adaptation to salinity stress, and here we show that gene gain and loss also play key roles in adaptation. We determined the extent of horizontal gene transfer (HGT) from prokaryotes and their role in the origin of novel functions in the Picochlorum clade. HGT is an ongoing and dynamic process in this algal clade with adaptation being driven by transfer, divergence, and loss. One HGT candidate that is differentially expressed under salinity stress is indolepyruvate decarboxylase that is involved in the production of a plant auxin that mediates bacteria-diatom symbiotic interactions. Large differences in levels of heterozygosity were found in diploid haplotypes among Picochlorum isolates. Biallelic divergence was pronounced in P. oklahomensis (salt plains environment) when compared with its closely related sister taxon Picochlorum SENEW3 (brackish water environment), suggesting a role of diverged alleles in response to environmental stress. Our results elucidate how microbial eukaryotes with limited gene inventories expand habitat range from mesophilic to halophilic through allelic diversity, and with minor but important contributions made by HGT. We also explore how the nature and quality of genome data may impact inference of nuclear ploidy.


September 21, 2019  |  

Phased diploid genome assembly with single-molecule real-time sequencing.

While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.


July 19, 2019  |  

High-quality assembly of an individual of Yoruban descent

De novo assembly of human genomes is now a tractable effort due in part to advances in sequencing and mapping technologies. We use PacBio single-molecule, real-time (SMRT) sequencing and BioNano genomic maps to construct the first de novo assembly of NA19240, a Yoruban individual from Africa. This chromosome-scaffolded assembly of 3.08 Gb with a contig N50 of 7.25 Mb and a scaffold N50 of 78.6 Mb represents one of the most contiguous high-quality human genomes. We utilize a BAC library derived from NA19240 DNA and novel haplotype-resolving sequencing technologies and algorithms to characterize regions of complex genomic architecture that are normally lost due to compression to a linear haploid assembly. Our results demonstrate that multiple technologies are still necessary for complete genomic representation, particularly in regions of highly identical segmental duplications. Additionally, we show that diploid assembly has utility in improving the quality of de novo human genome assemblies.


July 19, 2019  |  

The impact of third generation genomic technologies on plant genome assembly.

Since the introduction of next generation sequencing, plant genome assembly projects do not need to rely on dedicated research facilities or community-wide consortia anymore, even individual research groups can sequence and assemble the genomes they are interested in. However, such assemblies are typically not based on the entire breadth of genomic technologies including genetic and physical maps and their contiguities tend to be low compared to the full-length gold standard reference sequences. Recently emerging third generation genomic technologies like long-read sequencing or optical mapping promise to bridge this quality gap and enable simple and cost-effective solutions for chromosomal-level assemblies.


July 19, 2019  |  

Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation.

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes. Published by Cold Spring Harbor Laboratory Press.


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