Learn how Single Molecule, Real-Time (SMRT) Sequencing and the Sequel IIe System and will accelerate your research by delivering highly accurate long reads to provide the most comprehensive view of genomes, transcriptomes and epigenomes.
Structural variation accounts for much of the variation among human genomes. Structural variants of all types are known to cause Mendelian disease and contribute to complex disease. Learn how long-read sequencing is enabling detection of the full spectrum of structural variants to advance the study of human disease, evolution and genetic diversity.
The Agilent Femto Pulse system automated pulsed-field CE instrument is a fast, high-resolution benchtop capillary electrophoresis (CE) platform that utilizes pulsed-field electrophoresis to separate high molecular weight DNA fragments. This platform allows important DNA quality checkpoints to be completed in less than 1.5 hours with minimal sample input for de novo large genome sequencing projects and other PacBio applications leveraging multi-kilobase read lengths. The instrument can be used in place of gel-based pulsed-field electrophoresis (PFGE) systems to fully support generation of large-insert SMRTbell libraries with accurate sizing to 165 kb. Alternative DNA sizing instruments cannot accurately resolve large DNA fragments in this range.
Explore how high-quality genomes contribute to critical scientific endeavors.
Explore how highly accurate long-read sequencing enabled sequencing the large and highly complex California redwood genome.
At Cold Spring Harbor Laboratory, scientists used SMRT Sequencing to decode one of the most challenging cancer genomes ever encountered. Along the way, they built a portfolio of open-access analysis tools that will help researchers everywhere make structural variation discoveries with long-read sequencing data.
At DuPont Pioneer, DNA sequencing is paramount for R&D to reveal the genetic basis for traits of interest in commercial crops such as maize, soybean, sorghum, sunflower, alfalfa, canola, wheat, rice, and others. They cannot afford to wait the years it has historically taken for high-quality reference genomes to be produced. Nor can they rely on a single reference to represent the genetic diversity in its germplasm.
With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System, you can easily and cost effectively generate highly accurate long reads (HiFi reads, >99% single-molecule accuracy) from genes or regions of interest ranging in size from several hundred base pairs to 20 kb. Target all types of variation across relevant genomic regions, including low complexity regions like repeat expansions, promoters, and flanking regions of transposable elements.
In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties such as structure, function, or subcellular localization. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be underscored. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 – 10 kb, fully intact RNA molecules can be sequenced using SMRT Sequencing (avg. read length: 10-15 kb) without requiring fragmentation or post-sequencing assembly. Our open-source computational pipeline delivers high-quality, non-redundant sequences for unambiguous identification of alternative splicing events, alternative transcriptional start sites, polyA tail, and gene fusion events. The standard Iso-Seq protocol workflow available for all researchers is presented using a deep dataset of full- length cDNA sequences from the MCF-7 cancer cell line, and multiple tissues (brain, heart, and liver). Detected novel transcripts approaching 10 kb and alternative splicing events are highlighted. Even in extensively profiled samples, the method uncovered large numbers of novel alternatively spliced isoforms and previously unannotated genes.
Since the advent of Next-Generation Sequencing (NGS), the cost of de novo genome sequencing and assembly have dropped precipitately, which has spurred interest in genome sequencing overall. Unfortunately the contiguity of the NGS assembled sequences, as well as the accuracy of these assemblies have suffered. Additionally, most NGS de novo assemblies leave large portions of genomes unresolved, and repetitive regions are often collapsed. When compared to the reference quality genome sequences produced before the NGS era, the new sequences are highly fragmented and often prove to be difficult to properly annotate. In some cases the contiguous portions are smaller than the average gene size making the sequence not nearly as useful for biologists as the earlier reference quality genomes including of Human, Mouse, C. elegans, or Drosophila. Recently, new 3rd generation sequencing technologies, long-range molecular techniques, and new informatics tools have facilitated a return to high quality assembly. We will discuss the capabilities of the technologies and assess their impact on assembly projects across the tree of life from small microbial and fungal genomes through large plant and animal genomes. Beyond improvements to contiguity, we will focus on the additional biological insights that can be made with better assemblies, including more complete analysis genes in their flanking regulatory context, in-depth studies of transposable elements and other complex gene families, and long-range synteny analysis of entire chromosomes. We will also discuss the need for new algorithms for representing and analyzing collections of many complete genomes at once.
We show that linearizing and directly sequencing full-length fosmids simplifies the assembly problem such that it is possible to unambiguously assemble individual haplotypes for the highly repetitive 100-200 kb killer Ig-like receptor (KIR) gene loci of chromosome 19. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the KIR locus, means that sequence assembly of pooled samples into complete haplotypes is difficult and in many cases impossible. The current maximum read length generated by SMRT Sequencing exceeds the length of a 40 kb fosmid; it is therefore possible to span an entire fosmid in one sequencing read. Shearing, sequencing and assembling fosmids in a shotgun approach is prone to errors when the underlying sequence is highly repetitive. We show that it is possible to directly sequence linearized fosmids and generate a high-quality consensus by simple alignment, removing the need for an error-prone assembly step. The high-quality sequence of complete fosmids can then be tiled into full haplotypes. We demonstrate the method on DNA samples from a number of individuals and fully recover the sequence of both haplotypes from a pool of KIR fosmids. The ability to haplotype and sequence complex immunogenetic regions will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. This simple and robust approach can be scaled-up allowing a complex genomic region to be sequenced at a population level. We expect such sequencing to be valuable in disease association research.
Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever and organ failure, but mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for this viral tolerance are unclear. Recent publications highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient, and at least some EVEs are under purifying selection, suggesting they are beneficial to the host. To characterize EVE biogenesis in a tractable system, we sequenced the Ae. aegypti cell line, Aag2, to 58-fold coverage and present a de novo assembly of the genome. The assembly contains 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, consisting of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify EVEs in the genome homologous to a range of extant viruses, many of which cluster in these regions of repetitive DNA. The contiguous assembly allows for more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing.
Structural variants (genomic differences =50 base pairs) contribute to the evolution of organisms traits and human disease. Most structural variants (SVs) are too small to detect with array comparative genomic hybridization but too large to reliably discover with short-read DNA sequencing. Recent studies in human genomes show that PacBio SMRT Sequencing sensitively detects structural variants.
Targeted sequencing employing PCR amplification is a fundamental approach to studying human genetic disease. PacBio’s Sequel System and supporting products provide an end-to-end solution for amplicon sequencing, offering better performance to Sanger technology in accuracy, read length, throughput, and breadth of informative data. Sample multiplexing is supported with three barcoding options providing the flexibility to incorporate unique sample identifiers during target amplification or library preparation. Multiplexing is key to realizing the full capacity of the 1 million individual reactions per Sequel SMRT Cell. Two analysis workflows that can generate high-accuracy results support a wide range of amplicon sizes in two ranges from 250 bp to 3 kb and from 3 kb to >10 kb. The Circular Consensus Sequencing workflow results in high accuracy through intra-molecular consensus generation, while high accuracy for the Long Amplicon Analysis workflow is achieved by clustering of individual long reads from multiple reactions. Here we present workflows and results for single- molecule sequencing of amplicons for human genetic analysis.
Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.
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