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Product Note: SMRTbell express template prep 2.0 for microbial multiplexing

2019

Summary +

The SMRTbell Express Template Prep Kit 2.0 provides a streamlined, single-tube reaction strategy to generate SMRTbell libraries from 500 bp to >50 kb insert size targets to support large-insert genomic libraries, multiplexed microbial genomes and amplicon sequencing. With this new formulation, we have increased both the yield and efficiency of SMRTbell library preparation for SMRT Sequencing while further minimizing handling-induced DNA damage to retain the integrity of genomic DNA (gDNA). This product note highlights the key benefits, performance, and resources available for obtaining complete microbial genome assemblies with multiplexed sequencing. By using a single-tube, addition-only strategy, the streamlined workflow reduces the number of AMPure clean-up steps. This provides an opportunity to explore automation solutions for high-volume projects, while using as little as 1 ug of input DNA.

Application Note: Microbial multiplexing workflow on the Sequel System

2019

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 8 hours bench time. Starting with high-quality genomic DNA (gDNA), samples are sheared to approximately 12 kb distribution, ligated with barcoded overhang adapters, pooled at equimolar representation, and sequenced. Demultiplexing of samples is automated, allowing for immediate genome assembly on our SMRT Link analysis software solution.

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

2019

Summary +

The Agilent Femto Pulse 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 in this range.

Product Note: SMRTbell express template prep 2.0 for large-Insert libraries

2019

Summary +

The SMRTbell Express Template Prep Kit 2.0 provides a streamlined, single-tube reaction strategy to generate SMRTbell libraries from 500 bp to >50 kb insert size targets to support large-insert genomic libraries, multiplexed microbial genomes and amplicon sequencing. With this new formulation, we have increased both the yield and efficiency of SMRTbell library preparation for SMRT Sequencing while further minimizing handling-induced DNA damage to retain the integrity of genomic DNA (gDNA). This product note highlights the key benefits, performance, and resources available for supporting de novo genome sequencing and structural variant detection projects. Our large-insert gDNA protocol has been streamlined to support SMRTbell library generation in only 4 hours, making complete construction in less than a day. This significantly reduces time to results for generating high-quality genome assemblies to fully characterize SNPs and structural variants. Additional key benefits of this template preparation kit and updated protocol include library generation with as little as 2 to 3 µg input gDNA and flexibility to accommodate and adjust input amount in accordance with the extracted gDNA quality.

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

2019

Summary +

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.

Application Brochure: Microbial communities – Complex microbial populations simplified

2018

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.

SMRT Analysis Brochure: Gain a deeper understanding of your sequencing data

2018

Summary +

The PacBio Platform includes an extensive software portfolio that employs key advantages of SMRT (Single Molecule, Real-Time) Sequencing technology: extraordinarily long reads, highest consensus accuracy, uniform coverage and simultaneous epigenetic characterization. Core elements of our analytical portfolio include SMRT Analysis software, DevNet and SMRT Compatible products.

Application Brief: Large genome whole genome sequencing – Best Practices

2018

Summary +

Single Molecule, Real-Time (SMRT) Sequencing on the Sequel System enables easy and affordable generation of high- quality de novo assemblies of even the most complex plant and animal genomes. With megabase-size contig N50s, consensus accuracies >99.99%, and tools for phasing haplotypes you can capture undetected SNVs, fully intact genes, and regulatory regions embedded in complex structures that fragmented draft genomes often miss.

Application Brief: Targeted sequencing for amplicons – Best Practices

2018

Summary +

With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System, you can easily and cost effectively generate high-fidelity, long reads (>99% single-molecule read accuracy) from genes or regions of interest ranging in size from several hundred base pairs to 20 kb. Target all types of variation across relevant genomic regions, including low complexity regions like repeat expansions, promoters, and flanking regions of transposable elements.

Application Brief: Long-read RNA sequencing – Best Practices

2018

Summary +

With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System, you can easily and affordably sequence transcript isoforms of up to 10 kb in their entirety. The Iso-Seq method allows users to generate full-length cDNA sequences — with no assembly required — in order to confidently characterize the full complement of transcript isoforms within targeted genes, or across an entire transcriptome.

Application Brief: Low-coverage, long-read whole genome sequencing for structural variation – Best Practices

2018

Summary +

With the Sequel System, you can affordably and sensitively characterize structural variation (SV) of all types ranging in size from tens to thousands of base pairs. Low-coverage, long-read whole genome sequencing (WGS) data provides rapid discovery of common SVs for population genetics studies and resolves rare SVs unique to an individual, with a very low false-discovery rate.