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Explore scientific publications featuring PacBio long-read sequencing data

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Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Human Mutation
ePub ahead of print

2018

Abstract +

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with Single Molecule, Real-Time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to eleven diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

High-resolution comparative analysis of great ape genomes.

Science
360

2018

Abstract +

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. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Long-read sequencing and de novo genome assembly of Ammopiptanthus nanus, a desert shrub.

GigaScience
ePub ahead of print

2018

Abstract +

Ammopiptanthus nanus is a rare broad-leaved shrub in the desert and arid regions of Central Asia. This plant species exhibits extremely high tolerance to drought and freezing and has been used in abiotic tolerance research in plants. As a relic of the Tertiary period, A. nanus is of great significance to plant biogeographic research in the ancient Mediterranean region. Here we report a draft genome assembly using the PacBio platform and gene annotation for A. nanus.A total of 64.72 gigabases (Gb) of raw PacBio Sequel reads were generated from four 20 kb libraries. After filtering, 64.53 Gb of clean reads were obtained, giving 72.59 × coverage depth. Assembly using Canu gave an assembly length of 823.74 Mb, with a contig N50 of 2.76 Mb. The final size of the assembled A. nanus genome was close to the 889 Mb estimated by k-mer analysis. The gene annotation completeness was evaluated by BUSCO, and 1,327 out of the 1,440 conserved genes (92.15%) could be found in the A. nanus assembly. Genome annotation revealed that 74.08% of the A. nanus genome is composed of repetitive elements, and 53.44% of long terminal repeat elements (LTRs). We predicted 37,188 protein-coding genes, of which 96.53% were functionally annotated.The genomic sequences of A. nanus could be a valuable source for comparative genomic analysis in the legume family, and will be useful for understanding the phylogenetic relationships of the Thermopsideae and the evolutionary response of plant species to the Qinghai Tibetan Plateau uplift.

Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications.

BMC Plant Biology
18, 125

2018

Abstract +

Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date.Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA3, suggesting that post-transcriptional regulation might be also involved in the GA3 response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA3-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition.This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.

FALCON-Phase: Integrating PacBio and Hi-C data for phased diploid genomes

BioRxiv
Preprint

2018

Abstract +

De novo genome assembly of outbred diploid organisms remains a challenge in computational biology due to the difficulty of resolving similar haplotypes. FALCON-Unzip, a phased diploid genome assembler, separates PacBio long-reads by haplotype during assembly. The assembler outputs contiguous primary contigs, which are pseudohaplotypes containing phased haplotype regions and collapsed haplotypes. The ability to phase depends on the density of heterozygous variants, depth of coverage, and read length. As a result, haplotype phase information is lost when phase blocks are interrupted by regions of low heterozygosity, resulting in phase switches. Here, we present FALCON-Phase, a new method that resolves phase-switches by reconstructing contig-length phase blocks using Hi-C short-reads mapped to both homozygous regions and phase blocks. Such Hi-C data contain ultra-long-range phasing information (>1Mb). The novel FALCON-Phase algorithm is highly accurate (>96%) when benchmarked against a pedigree-based truth-set. The FALCON-Phase pipeline can also be extended to scaffolds to generate chromosome-scale phase blocks. The code is freely available (https://github.com/phasegenomics/FALCON-Phase) under a BSD and attribution license.

Long reads: their purpose and place.

Human Molecular Genetics
ePub ahead of print

2018

Abstract +

In recent years long read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing (NGS), the cost of sequencing using long read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterising genomes at high resolution. In this article, we will endeavour to present an introduction to long read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful; and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

CRISPR-Cas9 interference in cassava linked to the evolution of editing-resistant geminiviruses

BioRxiv
Preprint

2018

Abstract +

We used CRISPR-Cas9 in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus (a member of a widespread and important family of plant-pathogenic DNA viruses) by cleaving the virustextquoteright replicative genome. We found that between 33 and 48% of edited virus genomes evolved a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. Our study highlights the potential for virus escape from this technology. Care should be taken to design CRISPR-Cas9 experiments that minimize the risk of virus escape.

A survey of transcriptome complexity in Sus scrofa using single-molecule long-read sequencing.

DNA Research
ePub ahead of print

2018

Abstract +

Alternative splicing (AS) and fusion transcripts produce a vast expansion of transcriptomes and proteomes diversity. However, the reliability of these events and the extend of epigenetic mechanisms have not been adequately addressed due to its limitation of uncertainties about the complete structure of mRNA. Here we combined single-molecule real-time sequencing, Illumina RNA-seq and DNA methylation data to characterize the landscapes of DNA methylation on AS, fusion isoforms formation and lncRNA feature and further to unveil the transcriptome complexity of pig. Our analysis identified an unprecedented scale of high-quality full-length isoforms with over 28,127 novel isoforms from 26,881 novel genes. More than 92,000 novel AS events were detected and intron retention predominated in AS model, followed by exon skipping. Interestingly, we found that DNA methylation played an important role in generating various AS isoforms by regulating splicing sites, promoter regions and first exons. Furthermore, we identified a large of fusion transcripts and novel lncRNAs, and found that DNA methylation of the promoter and gene body could regulate lncRNA expression. Our results significantly improved existed gene models of pig and unveiled that pig AS and epigenetic modify were more complex than previously thought.

Analysis of transcripts and splice isoforms in Red Clover (Trifolium pratense L.) by single-molecule long-read sequencing

BioRxiv
Preprint

2018

Abstract +

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2,194 novel isoforms from novel genes were identified. A total of 5,492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8,719 including 1,86,517 transcripts have at least one poly(A) site. Furthermore, we identified 4,333 long non-coding RNAs and 3,762 fusion transcripts. Our results show the feasibility of deep sequencing full-length RNA from red clover transcriptome on a single-molecule level.

Antigenic variation in the lyme spirochete: Insights into recombinational switching with a suggested role for error-prone repair.

Cell Reports
23, 2595-2605

2018

Abstract +

The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host's acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA's dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5' end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia

BioRxiv
Preprint

2018

Abstract +

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect over 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a novel human-specific tandem repeat in this region that is composed of 30 bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution, and may modulate neuropsychiatric disease risk in modern human populations.

Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.

Nature Genetics
50, 708-717

2018

Abstract +

To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.

Genome sequence of the progenitor of wheat A subgenome Triticum urartu.

Nature
ePub ahead of print

2018

Abstract +

Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid (Triticum turgidum, AABB) and hexaploid (Triticum aestivum, AABBDD) wheat1,2. Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping4,5. We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147 T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement.

Genomic variation in 3,010 diverse accessions of Asian cultivated rice.

Nature
557, 43-49

2018

Abstract +

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

High throughput single cell sequencing of both T-cell-receptor-beta alleles

BioRxiv
Preprint

2018

Abstract +

Allelic exclusion is a vital mechanism for the generation of monospecificity to foreign antigens in B- and T-lymphocytes. Here we developed a high-throughput barcoded method to simultaneously analyze the VDJ recombination status of both T cell receptor beta alleles in hundreds of single cells using Next Generation Sequencing.

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American Society of Human Genetics (ASHG) 2018

October 16, 2018-October 20, 2018

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