May 16, 2022  |  AAV

Application Brochure AAV Sequencing with HiFi Reads

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

August 19, 2021  |  AAV

Application Brochure: Gene editing validation with HiFi reads

With highly accurate long reads (HiFi reads) from the Sequel IIe System, powered by Single Molecule, Real-Time (SMRT) Sequencing technology, you can efficiently and cost effectively validate gene editing techniques including adeno-associated virus (AAV) and CRISPR-Cas9 approaches.

July 19, 2019  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats. We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntingtontextquoterights disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.

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