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Explore scientific publications featuring PacBio long-read sequencing data

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Mycobiome diversity: high-throughput sequencing and identification of fungi.

Nature reviews. Microbiology
ePub ahead of print

2018

Abstract +

Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous - and largely uncharted - taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.

Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay.

PLoS genetics
14, e1007807

2018

Abstract +

Chardonnay is the basis of some of the world's most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding.

The genome of common long-arm octopus Octopus minor.

GigaScience
7

2018

Abstract +

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.

De novo assembly of haplotype-resolved genomes with trio binning.

Nature biotechnology
ePub ahead of print

2018

Abstract +

Complex allelic variation hampers the assembly of haplotype-resolved sequences from diploid genomes. We developed trio binning, an approach that simplifies haplotype assembly by resolving allelic variation before assembly. In contrast with prior approaches, the effectiveness of our method improved with increasing heterozygosity. Trio binning uses short reads from two parental genomes to first partition long reads from an offspring into haplotype-specific sets. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. We used trio binning to recover both haplotypes of a diploid human genome and identified complex structural variants missed by alternative approaches. We sequenced an F1 cross between the cattle subspecies Bos taurus taurus and Bos taurus indicus and completely assembled both parental haplotypes with NG50 haplotig sizes of >20 Mb and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We suggest that trio binning improves diploid genome assembly and will facilitate new studies of haplotype variation and inheritance.

Reference grade characterization of polymorphisms in full-length HLA class I and II genes with short-read sequencing on the Ion PGM system and long-reads generated by Single Molecule, Real-time Sequencing on the PacBio platform

Frontiers in immunology
9, 2294

2018

Abstract +

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field.

Nucleic acids research
ePub ahead of print

2018

Abstract +

We present a new method, CIDER-Seq (Circular DNA Enrichment sequencing) for the unbiased enrichment and long-read sequencing of viral-sized circular DNA molecules. We used CIDER-Seq to produce single-read full-length virus genomes for the first time. CIDER-Seq combines PCR-free virus enrichment with Single Molecule Real Time sequencing and a new sequence de-concatenation algorithm. We apply our technique to produce >1200 full-length, highly accurate geminivirus genomes from RNAi-transgenic and control plants in a field trial in Kenya. Using CIDER-Seq we can demonstrate for the first time that the expression of antiviral double-stranded RNA (dsRNA) in transgenic plants causes a consistent shift in virus populations towards species sharing low homology to the transgene derived dsRNA. Our method and its application in an economically important crop plant opens new possibilities in periodic virus sequence surveillance and accurate profiling of diverse circular DNA elements.

Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L.

Nature genetics
50, 1565-1573

2018

Abstract +

Modern sugarcanes are polyploid interspecific hybrids, combining high sugar content from Saccharum officinarum with hardiness, disease resistance and ratooning of Saccharum spontaneum. Sequencing of a haploid S. spontaneum, AP85-441, facilitated the assembly of 32 pseudo-chromosomes comprising 8 homologous groups of 4 members each, bearing 35,525 genes with alleles defined. The reduction of basic chromosome number from 10 to 8 in S. spontaneum was caused by fissions of 2 ancestral chromosomes followed by translocations to 4 chromosomes. Surprisingly, 80% of nucleotide binding site-encoding genes associated with disease resistance are located in 4 rearranged chromosomes and 51% of those in rearranged regions. Resequencing of 64 S. spontaneum genomes identified balancing selection in rearranged regions, maintaining their diversity. Introgressed S. spontaneum chromosomes in modern sugarcanes are randomly distributed in AP85-441 genome, indicating random recombination among homologs in different S. spontaneum accessions. The allele-defined Saccharum genome offers new knowledge and resources to accelerate sugarcane improvement.

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections

BioRxiv
Preprint

2018

Abstract +

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 alleles. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving allelic differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads - this was observed as early as day 3, the earliest that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse allele expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and identifying mosaic gene formation unprecedentedly early during the infection process.

Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice.

Nature biotechnology
36, 839-842

2018

Abstract +

We report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.

De novo assembly of two Swedish genomes reveals missing segments from the human GRCh38 reference and improves variant calling of population-scale sequencing data.

Genes
9

2018

Abstract +

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Elevated expression of a minor isoform of ANK3 is a risk factor for bipolar disorder.

Translational Psychiatry
8, 210

2018

Abstract +

Ankyrin-3 (ANK3) is one of the few genes that have been consistently identified as associated with bipolar disorder by multiple genome-wide association studies. However, the exact molecular basis of the association remains unknown. A rare loss-of-function splice-site SNP (rs41283526*G) in a minor isoform of ANK3 (incorporating exon ENSE00001786716) was recently identified as protective of bipolar disorder and schizophrenia. This suggests that an elevated expression of this isoform may be involved in the etiology of the disorders. In this study, we used novel approaches and data sets to test this hypothesis. First, we strengthen the statistical evidence supporting the allelic association by replicating the protective effect of the minor allele of rs41283526 in three additional large independent samples (meta-analysis p-values: 6.8E-05 for bipolar disorder and 8.2E-04 for schizophrenia). Second, we confirm the hypothesis that both bipolar and schizophrenia patients have a significantly higher expression of this isoform than controls (p-values: 3.3E-05 for schizophrenia and 9.8E-04 for bipolar type I). Third, we determine the transcription start site for this minor isoform by Pacific Biosciences sequencing of full-length cDNA and show that it is primarily expressed in the corpus callosum. Finally, we combine genotype and expression data from a large Norwegian sample of psychiatric patients and controls, and show that the risk alleles in ANK3 identified by bipolar disorder GWAS are located near the transcription start site of this isoform and are significantly associated with its elevated expression. Together, these results point to the likely molecular mechanism underlying ANK3´s association with bipolar disorder.

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

GigaScience
7

2018

Abstract +

In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the whole-genome and transcriptome sequencing, followed by subsequent de novo assembly, of the E. spelaea genome solely using the Pacific Biosciences (PacBio) long-read sequencing platform.The newly assembled E. spelaea genome is 1.97 Gb in length and consists of 4,470 sequences with a contig N50 of 8.0 Mb. Identified repeat elements covered 34.65% of the genome, and 20,640 unique protein-coding genes with 39,526 transcripts were annotated.We demonstrated that the PacBio long-read sequencing platform alone is sufficient to generate a comprehensive de novo assembled genome and transcriptome of an important bat species. These results will provide useful insights and act as a resource to expand our understanding of bat evolution, ecology, physiology, immunology, viral infection, and transmission dynamics.

Extreme resistance to Potato Virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor

BioRxiv
Preprint

2018

Abstract +

Potato virus Y (PVY) is a major potato pathogen that causes annual losses of billions of dollars. Control of its transmission requires extensive use of environmentally damaging insecticides. Rysto confers extreme resistance (ER) to PVY and is a valuable trait in resistance breeding programs. We isolated Rysto using Resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences Single-Molecule Real Time Sequencing). Rysto encodes a nucleotide binding-leucine rich repeat (NLR) protein with an N-terminal TIR domain, and is sufficient for PVY perception and extreme resistance in transgenic potato plants. We investigated the requirements for Rysto-dependent extreme resistance, and showed that Rysto function is temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

eLife
7

2018

Abstract +

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.© 2018, Fallon et al.

Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Nature
ePub ahead of print

2018

Abstract +

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.

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Advances in Genome Biology and Technology (AGBT) 2019

February 27, 2019-March 2, 2019

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