In this technical application note we demonstrate how long reads can be used to study hard-to-sequence regions. We will give an overview of the methods and considerations when targeting genomic regions or transcript sequencing and present various results and examples of applications.
PacBio offers end-to-end barcoding workflows for multiplexing amplicons up to 10 kb.
The Targeted Locus Amplification (TLA) Technology from Cergentis enables the targeted, hypothesis-neutral, amplification of any genomic locus of interest over 50 kb using just one primer pair complementary to a short locus-specific sequence. TLA is a strategy to selectively amplify complete loci on the basis of crosslinking physically proximal sequences. Unlike other targeted sequencing methods, TLA works without prior detailed locus information, as one primer pair is sufficient to amplify tens to hundreds of kilobases of DNA surrounding that locus. In a separate application of TLA, the unamplified template can be used for genome-wide phasing and assembly. TLA enables targeted…
Target enrichment capture methods allow scientists to rapidly interrogate important genomic regions of interest for variant discovery, including SNPs, gene isoforms, and structural variation. Custom targeted sequencing panels are important for characterizing heterogeneous, complex diseases and uncovering the genetic basis of inherited traits with more uniform coverage when compared to PCR-based strategies. With the increasing availability of high-quality reference genomes, customized gene panels are readily designed with high specificity to capture genomic regions of interest, thus enabling scientists to expand their research scope from a single individual to larger cohort studies or population-wide investigations. Coupled with PacBio long-read sequencing, these…
With Single Molecule, Real-Time (SMRT) Sequencing, you can affordably characterize complete microbial genomes. For most microbes, closed genomes and accessory plasmids can be assembled using PacBio generated data from a single library in a single run — with turn-around times as short as one day.
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 lso-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 trenscriptome.
With Single Molecule, Real—Time (SMRT) Sequencing and the Sequel System, you can easily and affordably generate high quality assemblies for even the most complex genomes. Users are regularly achieving plant and animal genome assemblies with megabase—size contig N50s and consensus accuracies >99.999%, resulting in the most complete genomes available today.
The Fragment Analyzer instrument is a fast, high-resolution benchtop capillary electrophoresis (CE) platform that utilizes proprietary markers to accurately size fragments ranging from 10 bp up to 50 kb. This platform allows important DNA quality checkpoints to be completed in 1 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, 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.