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Explore scientific publications featuring PacBio long-read sequencing data

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Megabase Length Hypermutation Accompanies Human Structural Variation at 17p11.2.

Cell
176, 1310-1324.e10

2019

Abstract +

DNA rearrangements resulting in human genome structural variants (SVs) are caused by diverse mutational mechanisms. We used long- and short-read sequencing technologies to investigate end products of de novo chromosome 17p11.2 rearrangements and query the molecular mechanisms underlying both recurrent and non-recurrent events. Evidence for an increased rate of clustered single-nucleotide variant (SNV) mutation in cis with non-recurrent rearrangements was found. Indel and SNV formation are associated with both copy-number gains and losses of 17p11.2, occur up to ~1 Mb away from the breakpoint junctions, and favor C > G transversion substitutions; results suggest that single-stranded DNA is formed during the genesis of the SV and provide compelling support for a microhomology-mediated break-induced replication (MMBIR) mechanism for SV formation. Our data show an additional mutational burden of MMBIR consisting of hypermutation confined to the locus and manifesting as SNVs and indels predominantly within genes. Copyright © 2019 Elsevier Inc. All rights reserved.

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Genes
10

2019

Abstract +

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

A 12-kb structural variation in progressive myoclonic epilepsy was newly identified by long-read whole-genome sequencing.

Journal of human genetics
ePub ahead of print

2019

Abstract +

We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions and three insertions) on the genes tagged with autosomal recessive phenotypes. Among them, a 12.4-kb deletion involving the CLN6 gene was the top candidate because its homozygous abnormalities cause neuronal ceroid lipofuscinosis. This deletion included the initiation codon and was found in a GC-rich region containing multiple repetitive elements. These results indicate the presence of a causal variant in a difficult-to-sequence region and suggest that such variants that remain enigmatic after the application of current whole-exome sequencing technology could be uncovered by unbiased application of long-read whole-genome sequencing.

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

BMC genomics
20, 120

2019

Abstract +

Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood.In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects.This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Scientific reports
9, 2709

2019

Abstract +

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea's unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat.

Genetics in medicine
ePub ahead of print

2019

Abstract +

To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1).We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n?=?11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis.CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (=50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length =91 repeats) indicating that expanded alleles behave dynamically.CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

Identification of a Xist silencing domain by Tiling CRISPR.

Scientific reports
9, 2408

2019

Abstract +

Despite essential roles played by long noncoding RNAs (lncRNAs) in development and disease, methods to determine lncRNA cis-elements are lacking. Here, we developed a screening method named "Tiling CRISPR" to identify lncRNA functional domains. Using this approach, we identified Xist A-Repeats as the silencing domain, an observation in agreement with published work, suggesting Tiling CRISPR feasibility. Mechanistic analysis suggested a novel function for Xist A-repeats in promoting Xist transcription. Overall, our method allows mapping of lncRNA functional domains in an unbiased and potentially high-throughput manner to facilitate the understanding of lncRNA functions.

Longitudinal HIV sequencing reveals reservoir expression leading to decay which is obscured by clonal expansion.

Nature communications
10, 728

2019

Abstract +

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.

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

BMC genomics
20, 119

2019

Abstract +

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.

Characterizing the major structural variant alleles of the human genome.

Cell
176, 663-675.e19

2019

Abstract +

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. Copyright © 2018 Elsevier Inc. All rights reserved.

A high-quality de novo genome assembly from a single mosquito using PacBio sequencing

Genes
10, 62

2019

Abstract +

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

bioRxiv
Preprint

2019

Abstract +

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular, Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3textquoteright UTR lengths, as well as novel 5textquoteright starts and 3textquoteright ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114kb SNCA region, with the longest phased block exceeding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

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

Nature communications
10, 260

2019

Abstract +

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).

Fast and accurate long-read assembly with wtdbg2

bioRxiv
Preprint

2019

Abstract +

Existing long-read assemblers require tens of thousands of CPU hours to assemble a human genome and are being outpaced by sequencing technologies in terms of both throughput and cost. We developed a novel long-read assembler wtdbg2 that, for human data, is tens of times faster than published tools while achieving comparable contiguity and accuracy. It represents a significant algorithmic advance and paves the way for population-scale long-read assembly in future.

Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

bioRxiv
Preprint

2019

Abstract +

The major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

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