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Tuesday, July 27, 2021

Infographic: SMRT Sequencing – How it works

PacBio Systems are powered by Single Molecule, Real-Time (SMRT) Sequencing, a technology proven to produce exceptionally long reads with high accuracy. SMRT Sequencing allows you to accelerate your science with the complete range of PacBio applications to produce data you can trust.

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Tuesday, June 1, 2021

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens.

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic…

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Tuesday, June 1, 2021

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence,…

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Tuesday, June 1, 2021

An improved circular consensus algorithm with an application to detection of HIV-1 Drug-Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger Sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach arbitrarily high quality across longer insert lengths at a lower cost and higher throughput than Sanger Sequencing. We apply this new algorithm, dubbed CCS2, to the characterization of the HIV-1 K103N drug-resistance associated mutation, which…

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Tuesday, June 1, 2021

Multiplexing strategies for microbial whole genome SMRT Sequencing

The increased throughput of the RS II and Sequel Systems enables multiple microbes to be sequenced on a single SMRT Cell. This multiplexing can be readily achieved by simply incorporating a unique barcode for each microbe into the SMRTbell adapters after shearing genomic DNA using a streamlined library construction process. Incorporating a barcode without the requirement for PCR amplification prevents the loss of epigenetic information (e.g., methylation signatures), and the generation of chimeric sequences, while the modified protocol eliminates the need to build several individual SMRTbell libraries. We multiplexed up to 8 unique strains of H. pylori. Each strain was…

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Tuesday, June 1, 2021

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.

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Tuesday, June 1, 2021

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over…

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Tuesday, June 1, 2021

No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method

Targeted sequencing of genomic DNA requires an enrichment method to generate detectable amounts of sequencing products. Genomic regions with extreme composition bias and repetitive sequences can pose a significant enrichment challenge. Many genetic diseases caused by repeat element expansions are representative of these challenging enrichment targets. PCR amplification, used either alone or in combination with a hybridization capture method, is a common approach for target enrichment. While PCR amplification can be used successfully with genomic regions of moderate to high complexity, it is the low-complexity regions and regions containing repetitive elements sometimes of indeterminate lengths due to repeat expansions that…

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Friday, February 5, 2021

i5K Webinar: High-quality de novo insect genome assemblies using PacBio sequencing

PacBio Sequencing is characterized by very long sequence reads (averaging > 10,000 bases), lack of GC-bias, and high consensus accuracy. These features have allowed the method to provide a new gold standard in de novo genome assemblies, producing highly contiguous (contig N50 > 1 Mb) and accurate (> QV 50) genome assemblies. We will briefly describe the technology and then highlight the full workflow, from sample preparation through sequencing to data analysis, on examples of insect genome assemblies, and illustrate the difference these high-quality genomes represent with regard to biological insights, compared to fragmented draft assemblies generated by short-read sequencing.

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Friday, February 5, 2021

Webinar: Using Iso-Seq analysis to build a better annotation

Long-read sequencing technologies like Iso-Seq analysis present researchers with a powerful tool for probing the transcriptomes of many species. The ability to sequence transcripts from end-to-end has revealed transcription complexity on a scale that was previously impossible. This sequence rich information has also improved our ability to predict transcript functions and biotypes. Researchers can now use Iso-Seq analysis to discover transcript models in almost any species with an accuracy on par with human and mouse annotations. In this webinar, Richard Kuo discusses the core concepts behind Iso-Seq analysis and how to use it to improve or build a new transcriptome…

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Friday, February 5, 2021

Webinar: Sequence with Confidence – Introducing the Sequel II System

In this webinar, Jonas Korlach, Chief Scientific Officer, PacBio provides an overview of the features and the advantages of the new Sequel II System. Kiran Garimella, Senior Computational Scientist, Broad Institute of MIT and Harvard University, describes his work sequencing humans with HiFi reads enabling discovery of structural variants undetectable in short reads. Luke Tallon, Scientific Director, Genomics Resource Center, Institute for Genome Sciences, University of Maryland School of Medicine, covers the GRC’s work on bacterial multiplexing, 16S microbiome profiling, and shotgun metagenomics. Finally, Shane McCarthy, Senior Research Associate, University of Cambridge, focuses on the scaling and affordability of high-quality…

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