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Thursday, August 19, 2021

Whitepaper: Structural variation in the human genome

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.

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Thursday, August 19, 2021

Product Note: Fast, high-resolution DNA sizing with the fragment analyzer system

The Agilent 5200, 5300, and 5400 Fragment Analyzer instruments are fast, high-resolution benchtop capillary electrophoresis (CE) platforms that utilize proprietary markers to accurately size fragments ranging from 10 to 50 kb. This platform allows important DNA quality checkpoints to be completed in one hour for de novo large-genome sequencing projects and other PacBio applications leveraging multi-kilobase read lengths. The instrument can be used in place of time-consuming QC steps involving pulsed field gel electrophoresis (PFGE), saving time by avoiding multiple overnight gel runs when preparing large-insert SMRTbell libraries. Alternative DNA-sizing instruments cannot accurately resolve large DNA fragments in this range.

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Thursday, August 19, 2021

Product Note: Fast, high-resolution DNA sizing with the Agilent Femto Pulse system

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…

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Thursday, August 19, 2021

Application Note: Microbial multiplexing workflow on the Sequel System

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 8 hours bench time. Starting with high-quality genomic DNA (gDNA),…

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Thursday, August 19, 2021

Case Study: Pioneering a pan-genome reference collection

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.

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Thursday, August 19, 2021

Case Study: Diving Deep – Revealing the mysteries of marine life with SMRT Sequencing

Many scientists are using PacBio Single Molecule, Real-Time (SMRT) Sequencing to explore the genomes and transcriptomes of a wide variety of marine species and ecosystems. These studies are already adding to our understanding of how marine species adapt and evolve, contributing to conservation efforts, and informing how we can optimize food production through efficient aquaculture.

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Thursday, August 19, 2021

Application Brief: Variant detection using whole genome sequencing with HiFi reads – Best Practices

With highly accurate long reads (HiFi reads) from the Sequel II or IIe Systems you can comprehensively detect variants in 100s to 1000s of genomes in a year. HiFi reads provide high precision and recall for single nucleotide variants (SNVs), indels, structural variants (SVs), and copy number variants (CNVs), including in difficult-to-map repetitive regions.

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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

Genomic Architecture of the KIR and MHC-B and -C Regions in Orangutan

PacBio 2013 User Group Meeting Presentation Slides: Lisbeth Guethlein from Stanford University School of Medicine looked at highly repetitive and variable immune regions of the orangutan genome. Guethlein reported that “PacBio managed to accomplish in a week what I have been working on for a couple years” (with Sanger sequencing), and the results were concordant. “Long story short, I was a happy customer.”

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Tuesday, June 1, 2021

Getting the most out of your PacBio libraries with size selection.

PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated…

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Tuesday, June 1, 2021

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark…

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