PacBio Literature

Extract HMW DNA for HiFi sequencing for one-stop sample-to-answer workflow and support

The HiFiViral SARS-CoV-2 kit is an end-to-end solution for genomic surveillance of COVID-19 variants. The kit utilizes molecular inversion probes (MIPs), a differentiated enrichment technology which combines the simplicity and cost-effectiveness of PCR with the resilience to mutations of target capture. The MIPs approach, coupled with PacBio HiFi reads and SMRT® Link software analysis, delivers several advantages over PCR amplicon sequencing

AMPure® PB bead size selection is a simple, quick, and scalable approach to deplete molecules less than 5 kb in a SMRTbell® library. For high-quality DNA, the sequencing performance and application metrics for assembly and variant calling are comparable to gel cassette size-selection methods.

With HiFi target enrichment you can easily and cost-effectively sequence just the genes you care about at scale. Design custom gene panels with our partners at Twist, sequence them with long and accurate HiFi reads, and get all the benefits you expect from HiFi reads: comprehensive detection of single nucleotide variants, structural variants, and indels. HiFi target enrichment is the only technology that can resolve complex gene families like HLA or Cytochrome P450 genes with haplotype resolution.​

Bacteria, viruses, and fungi all affect agriculturally important plant and animal species. Accurate HiFi sequencing allows researchers to study microbes and microbial communities in high resolution. Understanding how microbes and microbial communities can impact the health of plants and livestock allows scientists to maximize food production and quality and protect environmental ecosystems.

Accurate and flexible HiFi solutions for metagenomics and microbiome sequencing allow researchers to study microbes and microbial communities in high resolution without the need for culturing.

Scale your whole genome sequencing (WGS) and epigenome workflows with PacBio® HiFi reads employing a single technician working one day a week. Sequence 25 human genomes at ≥ 30-fold coverage each month using four Sequel® II systems.*

With Single Molecule, Real-Time (SMRT®) sequencing on the Sequel® IIe systems, you can sequence AAV genome populations to identify truncation, mutation, and host integration events. The AAV workflow from PacBio® accommodates both scAAV and ssAAV constructs with easy on-instrument HiFi read generation

Detect, track, and characterize pathogens to a new standard of completeness and precision. PacBio® solutions offer unmatched accuracy and long read lengths to support a wide range of pathogen surveillance applications.

PacBio recommends to use size-selection beads are to remove short DNA fragments and enrich for the long fragments that are most valuable for downstream analysis. Although size-selection beads have many advantages, alternative methods — like gels and DNA precipitation — offer more flexibility in defining a size cutoff. This technical note provides guidance for alternative methods of size selection of WGS libraries prepared with the SMRTbell prep kit 3.0.

HiFi sequencing produces long, accurate reads of the 4 DNA bases — A, C, G, and T — that deliver the most comprehensive characterization of genomes. But HiFi sequencing is not limited to characterizing the genome. It simultaneously measures the epigenome by detecting a fifth base — 5mC at CpG sites — without requiring special library preparation like bisulfite treatment. This detects distinct regional epigenetic patterns, accesses methylation in the full genome, and identifies allele-specific methylation.

PacBio recommends that DNA shearing for whole genome sequencing be performed on the Diagenode Megaruptor 3 system, which is the only system validated by PacBio for shearing, in order to produce consistent HiFi yield and quality. When a Megaruptor 3 system is unavailable, the Covaris g-TUBE presents an alternative shearing method that does not require instrumentation beyond a standard microcentrifuge. This Technical note provides guidelines for using the Covaris g-TUBE for DNA shearing.

Sample preparation is a critical factor for whole genome sequencing (WGS) projects that impacts sequencing yield, read length, and ultimately performance for variant calling and genome assembly. Best practices to optimize performance for PacBio HiFi sequencing are to collect whole human blood, store with potassium EDTA as the anticoagulant for fewer than 2 days at 4°C, and extract DNA from 200 μL of blood using the Nanobind CBB Big DNA kit. When it is not possible to extract from fresh samples, storing samples at 4°C maximizes extraction yield and minimizes degradation for at least 9 days.

The Iso-Seq method utilizes long-read transcript sequencing to reliably capture full-length transcript isoforms without the need for computational assembly. PacBio’s long-read RNA sequencing is also significantly less error-prone than other long-read sequencing technologies, offering more robust and accurate isoform discovery power.9,10 Future research will continue to explore the complex and mostly unexplored landscape of isoforms across cancer types

HiFiViral for SARS-CoV-2 is a simple-to-use, scalable, cost-effective solution for sequencing the entire SARS-CoV-2 genome. This fully kitted solution uses a novel approach that is robust to new variants and comprehensively detects all types of mutations.