fbpx
X

Quality Statement

Pacific Biosciences is committed to providing high-quality products that meet customer expectations and comply with regulations. We will achieve these goals by adhering to and maintaining an effective quality-management system designed to ensure product quality, performance, and safety.

X

Image Use Agreement

By downloading, copying, or making any use of the images located on this website (“Site”) you acknowledge that you have read and understand, and agree to, the terms of this Image Usage Agreement, as well as the terms provided on the Legal Notices webpage, which together govern your use of the images as provided below. If you do not agree to such terms, do not download, copy or use the images in any way, unless you have written permission signed by an authorized Pacific Biosciences representative.

Subject to the terms of this Agreement and the terms provided on the Legal Notices webpage (to the extent they do not conflict with the terms of this Agreement), you may use the images on the Site solely for (a) editorial use by press and/or industry analysts, (b) in connection with a normal, peer-reviewed, scientific publication, book or presentation, or the like. You may not alter or modify any image, in whole or in part, for any reason. You may not use any image in a manner that misrepresents the associated Pacific Biosciences product, service or technology or any associated characteristics, data, or properties thereof. You also may not use any image in a manner that denotes some representation or warranty (express, implied or statutory) from Pacific Biosciences of the product, service or technology. The rights granted by this Agreement are personal to you and are not transferable by you to another party.

You, and not Pacific Biosciences, are responsible for your use of the images. You acknowledge and agree that any misuse of the images or breach of this Agreement will cause Pacific Biosciences irreparable harm. Pacific Biosciences is either an owner or licensee of the image, and not an agent for the owner. You agree to give Pacific Biosciences a credit line as follows: "Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA" and also include any other credits or acknowledgments noted by Pacific Biosciences. You must include any copyright notice originally included with the images on all copies.

IMAGES ARE PROVIDED BY Pacific Biosciences ON AN "AS-IS" BASIS. Pacific Biosciences DISCLAIMS ALL REPRESENTATIONS AND WARRANTIES, EXPRESS, IMPLIED OR STATUTORY, INCLUDING, BUT NOT LIMITED TO, NON-INFRINGEMENT, OWNERSHIP, MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL Pacific Biosciences BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, PUNITIVE, OR CONSEQUENTIAL DAMAGES OF ANY KIND WHATSOEVER WITH RESPECT TO THE IMAGES.

You agree that Pacific Biosciences may terminate your access to and use of the images located on the PacificBiosciences.com website at any time and without prior notice, if it considers you to have violated any of the terms of this Image Use Agreement. You agree to indemnify, defend and hold harmless Pacific Biosciences, its officers, directors, employees, agents, licensors, suppliers and any third party information providers to the Site from and against all losses, expenses, damages and costs, including reasonable attorneys' fees, resulting from any violation by you of the terms of this Image Use Agreement or Pacific Biosciences' termination of your access to or use of the Site. Termination will not affect Pacific Biosciences' rights or your obligations which accrued before the termination.

I have read and understand, and agree to, the Image Usage Agreement.

I disagree and would like to return to the Pacific Biosciences home page.

Pacific Biosciences
Contact:
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),…

Read More »

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,…

Read More »

Tuesday, June 1, 2021

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis.

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. A 2 kb SMRTbell library only requires a few ng of gDNA when carrier DNA is added to the library. The resulting libraries can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes…

Read More »

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…

Read More »

Tuesday, June 1, 2021

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. Amplification and sequencing of the evolutionarily conserved 16S rRNA gene is an efficient method of profiling communities. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost, and species-level classification is often not possible. PacBio SMRT Sequencing easily spans the entire 1.5 kb 16S gene in a single read, producing highly accurate single-molecule sequences that can improve the identification of individual species in a metapopulation.However,…

Read More »

Tuesday, June 1, 2021

An improved circular consensus algorithm with an application to detect 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 high quality across longer insert lengths at a lower cost and higher throughput than Sanger sequencing. We applied CCS2 to the characterization of the HIV-1 K103N drug-resistance associated mutation in both clonal and patient samples.…

Read More »

Tuesday, June 1, 2021

Workflow for processing high-throughput, Single Molecule, Real-Time Sequencing data for analyzing the microbiome of patients undergoing fecal microbiota transplantation

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500 bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be…

Read More »

Tuesday, June 1, 2021

Multiplexing strategies for microbial whole genome SMRT Sequencing

As the throughput of the PacBio Systems continues to increase, so has the desire to fully utilize SMRT Cell sequencing capacity to multiplex microbes for whole genome sequencing. Multiplexing is readily achieved by incorporating a unique barcode for each microbe into the SMRTbell adapters and using a streamlined library preparation process. Incorporating barcodes without PCR amplification prevents the loss of epigenetic information and the generation of chimeric sequences, while eliminating the need to generate separate SMRTbell libraries. We multiplexed the genomes of up to 8 unique strains of H. pylori. Each genome was sheared and processed through adapter ligation in…

Read More »

Tuesday, June 1, 2021

WGS SMRT Sequencing of patient samples from a fecal microbiota transplant trial

Fecal samples were obtained from human subjects in the first blinded, placebo-controlled trial to evaluate the efficacy and safety of fecal microbiota transplant (FMT) for treatment of recurrent C. difficile infection. Samples included pre-and post-FMT transplant, post-placebo transplant, and the donor control; samples were taken at 2 and 8 week post-FMT. Sequencing was done on the PacBio Sequel System, with the goal of obtaining high quality sequences covering whole genes or gene clusters, which will be used to better understand the relationship between the composition and functional capabilities of intestinal microbiomes and patient health. Methods: Samples were randomly sheared to…

Read More »

Tuesday, June 1, 2021

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome using long-read sequencing

Sequence-based estimation of genetic diversity of Plasmodium falciparum, the most lethal malarial parasite, has proved challenging due to a lack of a complete genomic assembly. The skewed AT-richness (~80.6% (A+T)) of its genome and the lack of technology to assemble highly polymorphic sub-telomeric regions that contain clonally variant, multigene virulence families (i.e. var and rifin) have confounded attempts using short-read NGS technologies. Using single molecule, real-time (SMRT) sequencing, we successfully compiled all 14 nuclear chromosomes of the P. falciparum genome from telomere-to-telomere in single contigs. Specifically, amplification-free sequencing generated reads of average length 12 kb, with =50% of the reads…

Read More »

Tuesday, June 1, 2021

Multiplexing strategies for microbial whole genome sequencing using the Sequel System

For microbial sequencing on the PacBio Sequel System, the current yield per SMRT Cell is in excess relative to project requirements. Multiplexing offers a viable solution; greatly increasing throughput, efficiency, and reducing costs per genome. This approach is achieved by incorporating a unique barcode for each microbial sample into the SMRTbell adapters and using a streamlined library preparation process. To demonstrate performance,12 unique barcodes assigned to B. subtilis and sequenced on a single SMRT Cell. To further demonstrate the applicability of this method, we multiplexed the genomes of 16 strains of H. pylori. Each DNA was sheared to 10 kb,…

Read More »

Tuesday, June 1, 2021

Multiplexed complete microbial genomes on the Sequel System

Microbes play an important role in nearly every part of our world, as they affect human health, our environment, agriculture, and aid in waste management. Complete closed genome sequences, which have become the gold standard with PacBio long-read sequencing, can be key to understanding microbial functional characteristics. However, input requirements, consumables costs, and the labor required to prepare and sequence a microbial genome have in the past put PacBio sequencing out of reach for some larger projects. We have developed a multiplexed library prep approach that is simple, fast, and cost-effective, and can produce 4 to 16 closed bacterial genomes…

Read More »

Tuesday, June 1, 2021

Single chromosomal genome assemblies on the Sequel System with Circulomics high molecular weight DNA extraction for microbes

Background: The Nanobind technology from Circulomics provides an elegant HMW DNA extraction solution for genome sequencing of Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of high molecular weight (HMW) DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be completed in 7 kb repeats. Fragment size was increased to ~14 kb, with some fragments >30 kb. Results: Here we present a demonstration of these capabilities using isolates relevant to high-throughput sequencing applications, including common foodborne pathogens (Shigella, Listeria, Salmonella), and species often seen in…

Read More »

Tuesday, June 1, 2021

High-resolution evaluation of gut microbiota associated with intestinal maturation in early preterm neonates

Leaky gut, or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to necrotizing enterocolitis in preterm neonates. We recently revealed intestinal barrier maturation was associated with exclusive breastfeeding, less antibiotic exposure, most importantly, altered composition of the gut microbiota. However, sequencing short regions of 16S rRNA gene amplicon failed to identify the specific bacterial groups associated with improved or aberrant intestinal permeability. In this study, we performed high-throughput amplicon sequencing of the full length 16S rRNA gene with single-nucleotide resolution for a cohort of 66 preterm neonates born at 24-33 weeks of gestation who had…

Read More »

Tuesday, June 1, 2021

Comparison of sequencing approaches applied to complex soil metagenomes to resolve proteins of interest

Background: Long-read sequencing presents several potential advantages for providing more complete gene profiling of metagenomic samples. Long reads can capture multiple genes in a single read, and longer reads typically result in assemblies with better contiguity, especially for higher abundance organisms. However, a major challenge with using long reads has been the higher cost per base, which may lead to insufficient coverage of low-abundance species. Additionally, lower single-pass accuracy can make gene discovery for low-abundance organisms difficult. Methods: To evaluate the pros and cons of long reads for metagenomics, we directly compared PacBio and Illumina sequencing on a soil-derived sample,…

Read More »

1 2 3 48

Subscribe for blog updates:

Archives

Stay
Current

Visit our blog »