SMRT resources
Explore our collection of resources and learn how scientists use SMRT Sequencing to advance their research.
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Explore our database of scientific publications featuring PacBio long-read sequencing data.
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PacBio
20180705
Description +
The Iso-Seq method enables the sequencing of transcript isoforms from the 5’ end to their poly-A tails, eliminating the need for transcript reconstruction and inference. This webinar provides a comprehensive guide to Iso-Seq method data analysis, bioinformatics, and review key applications.
High-throughput SMRT Sequencing of clinically relevant targets
2018
Abstract +
Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The Circular Consensus Sequencing (CCS) analysis pipeline produces QV 30 data from each single intra-molecular multi-pass polymerase read, making it a reliable solution for detecting minor variant alleles with frequencies as low as 1 %. Long Amplicon Analysis (LAA) makes use of insert spanning full-length subreads originating from multiple individual copies of the target to generate highly accurate and phased consensus sequences (>QV50), offering a unique advantage for imputation free allele segregation and haplotype phasing. Here we present workflows and results for a range of SMRT Sequencing clinical applications. Specifically, we illustrate how the flexible multiplexing options, simple sample preparation methods and new developments in data analysis tools offered by PacBio in support of Sequel System 5.1 can come together in a variety of experimental designs to enable applications as diverse as high throughput HLA typing, mitochondrial DNA sequencing and viral vector integrity profiling of recombinant adeno-associated viral genomes (rAAV).
Single chromosomal genome assemblies on the Sequel System with Circulomics high molecular weight DNA extraction for microbes
2018
Abstract +
Background: Recent developments with Nanobind technology from Circulomics provide an elegant high molecular weight (HMW) DNA extraction solution to sequencing genomes from Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of HMW DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be performed manually or through automation using common instruments and can be completed in less than 1 hour for most sample types. Methods: We have validated several critical workflow steps for generating high quality microbial genome assemblies in a high throughput environment in a new streamlined microbial multiplexing workflow, which enables high-volume and cost-effective sequencing of up to 16 microbe samples totaling 30 Mb in genome size on a single SMRT Cell 1M using a target of 10 kb shear. Results: Here we share data demonstrating these new capabilities using isolates relevant to high throughput sequencing applications, including Shigella, common foodborne pathogens (Listeria, Salmonella), and species often seen in hospital settings (Klebsiella, Staphylococcus). We consistently achieved complete de novo microbial assemblies in =5 chromosomal contigs with minimum quality scores of QV40 (99.99% accuracy) using data from a single SMRTbell library, run on a single SMRT Cell 1M with the PacBio Sequel System, and analyzed with newly streamlined SMRT Analysis assembly methods. Conclusions: This simplified workflow helps to accelerate microbial genome characterization, requiring fewer than 24 hours for sample preparation and 10 hours for data collection.
PacBio
20180608
Description +
This video provides an overview of the techniques and steps of preparing samples, DNA, and libraries for PacBio Single Molecule, Real-Time (SMRT) Sequencing to be used in de novo assembly projects. In this video, a PacBio scientist covers how to assess DNA quantity and purity, size-selection of DNA libraries, and provides and introduction to SMRT Sequencing, including the benefits of long-reads when generating high-quality genome assemblies.
PacBio
20180608
Description +
This video provides an overview of the techniques and steps of generating a de novo genome assembly with long-read sequencing data generated using PacBio Single Molecule, Real-Time (SMRT) Sequencing. In this video, a PacBio scientist covers the benefits of long reads when generating high-quality genome assemblies, the latest tools for creating assemblies, including HGAP, FALCON and FALCON-Unzip, how to polish and assess the quality of a genome assembly, and how to submit an assembly to NCBI.
PacBio
20180607
Description +
This webinar, presented by Nisha Pillai, provides an overview of bioinformatics approaches for PacBio Single Molecule, Real-Time (SMRT) Sequencing data and discusses the whole genome sequencing application including: assembly workflow designs, an overview analysis tools for de novo assembly of SMRT Sequencing data (HGAP4, FALCON & FALCON-Unzip), and finally best practices and case studies.
Population-scale discovery of structural variants with PacBio SMRT Sequencing
2018
Abstract +
Structural variants (SVs) – genomic differences =50 base pairs – are few by count compared to single nucleotide variants (SNVs) and indels but include most of the base pairs that differ between two humans.
Characterizing the pan-genome of maize with PacBio SMRT Sequencing
2018
Abstract +
Maize is an amazingly diverse crop. A study in 2005 demonstrated that half of the genome sequence and one-third of the gene content between two inbred lines of maize were not shared. This diversity, which is more than two orders of magnitude larger than the diversity found between humans and chimpanzees, highlights the inability of a single reference genome to represent the full pan-genome of maize and all its variants. Here we present and review several efforts to characterize the complete diversity within maize using the highly accurate long reads of PacBio Single Molecule, Real-Time (SMRT) Sequencing. These methods provide a framework for a pan-genomic approach that can be applied to studies of a wide variety of important crop species.
PacBio
20180529
Description +
This webinar highlights global initiatives currently underway to use Single Molecule, Real-Time (SMRT) Sequencing to de novo assemble genomes of individuals representing multiple ethnic populations, thereby extending the diversity of available human reference genomes. In their presentations, Tina Graves-Lindsay from Washington University and Adam Ameur from Uppsala University spoke about diploid assemblies, discovering novel sequence and improving diversity of the current human reference genome. Finally, Paul Peluso of PacBio presented data from the recent effort to sequence a Puerto Rican genome and shared a SMRT Sequencing technology roadmap showing the next several upgrades for the Sequel System.
2017
Summary +
Explore the types of human genomic variation and the diseases known to be caused by structural variants.
2018
Summary +
Due to technology limitations, repeat-expansion disorders have gone without the needed base-level resolution of the disease causative long repetitive elements. Enrichment of these hard-to-amplify genomic regions is now possible with our no-amplification (No-Amp) targeted sequencing method utilizing the CRISPR/Cas9 system.
Summary +
Dan Geraghty, a researcher at Fred Hutchinson Cancer Research Center and CEO of Scisco Genetics, has spent much of his career focused on the genetics of immune response. Recently he talked to Mendelspod host Theral Timpson as part of a series of podcasts on the rise of long-read sequencing.
Summary +
With Single Molecule, Real-Time (SMRT) Sequencing, you can affordably characterize complete microbial genomes. For most microbes, closed genomes and accessory plasmids can be assembled using PacBio data from single libraries in a single run.
2017
Summary +
The ability to identify and understand the functions of the complex microbial populations living in, on, and around us requires comprehensive characterization of each community member. Long reads, high accuracy, and single-molecule resolution make Single Molecule, Real-Time (SMRT) Sequencing ideal for full-length 16S rRNA sequencing, long-read metagenomic profiling, and shotgun metagenomic assembly.
Summary +
Obtaining microbial genomes with the highest accuracy and contiguity is extremely important when exploring the functional impact of genetic and epigenetic variants on a genome-wide scale. A comprehensive view of the bacterial genome, including genes, regulatory regions, IS elements, phage integration sites, and base modifications is vital to understanding key traits such as antibiotic resistance, virulence, and metabolism. SMRT Sequencing provides complete genomes, often assembled into a single contig. Our streamlined microbial multiplexing procedure for the Sequel System, from library preparation to genome assembly, can be completed with less than 12 hours bench time. Starting with high-quality genomic DNA (gDNA), samples are sheared to a 10 kb distribution, ligated with barcoded adapters, pooled at equimolar representation, and sequenced. Demultiplexing of samples is automated, allowing for immediate genome assembly on our SMRT Link analysis software solution. The workflow supports up to 16-plex of de novo microbial genomes where the total genome sums up to 30 Mb on each SMRT Cell. As microbial genomes and gDNA samples vary in genetic complexity and quality, respectively, we describe general recommendations and best practices.
Summary +
The Targeted Locus Amplification (TLA) Technology from Cergentis enables the targeted, hypothesis-neutral, amplification of any genomic locus of interest over 50 kb using just one primer pair complementary to a short locus-specific sequence. TLA is a strategy to selectively amplify complete loci on the basis of crosslinking physically proximal sequences. Unlike other targeted sequencing methods, TLA works without prior detailed locus information, as one primer pair is sufficient to amplify tens to hundreds of kilobases of DNA surrounding that locus. In a separate application of TLA, the unamplified template can be used for genome-wide phasing and assembly. TLA enables targeted sequencing and detection of single nucleotide and structural variants in genes or regions of interest. SMRT sequencing enables end-to-end sequencing of multi-kilobase TLA amplicons or unamplified TLA templates. The combination of Cergentis' TLA and SMRT Sequencing technologies allows for sequencing and haplotyping of individual genes, chromosomes and genomes.
2016
Summary +
Single Molecule, Real-Time (SMRT) Sequencing directly detects DNA modifications by measuring variation in the polymerase kinetics of DNA base incorporation during sequencing. With high throughput, long reads, and the sensitivity to detect epigenetic modification without amplification or chemical conversions, the PacBio Systems offer scalable solutions for assessing DNA modifications in bacterial and eukaryotic genomes.
2017
Summary +
To bring precision medicine to every patient, cancer researchers need a more comprehensive view of all the somatic variants in genes, transcripts and whole genomes that drive cancer biology. Single Molecule, Real-Time (SMRT) Sequencing delivers the read lengths, uniform coverage, and accuracy needed to access the complete size spectrum of driver mutations — from rare single nucleotide variants to complex structural variants. Full-length transcript sequencing brings clarity to tumor-specific isoform and splice variant expression, enabling the discovery of novel biomarkers for early detection, tumor stratification, treatment response, and drug resistance. With SMRT Sequencing, scientists gain new insight into the most pressing questions in cancer research.
Summary +
PacBio offers end-to-end barcoding workflows for multiplexing amplicons up to 10 kb.
Summary +
DNA sequencing has been a critical tool for the field of microbiology since the technology was first invented. Here, we look at how advances in sequencing provide microbiologists the most comprehensive and cost-effective view of their research subjects.