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Wednesday, January 6, 2021

Video Poster: A new approach to Thalassemia and Ataxia carrier screening panels using CRISPR-Cas9 enrichment and long-read sequencing

Although PCR is a cost-effective way to enrich for genomic regions of interest for DNA sequencing, amplifying regions with extreme GC-content and long stretches of short tandem repeat (STR) sequences is often problematic and prone to sequence artifacts. This is especially true when developing multiplexed PCR assays for clinical applications such as carrier screening for multiple genes. The additional challenge is that all PCR primer pairs must be carefully selected to be compatible based on amplicon size and PCR conditions. Due to these experimental design constraints, a single tube with a high number of multiplexed PCR amplicons is difficult to…

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Wednesday, January 6, 2021

Webinar: SMRT Sequencing applications for human genomics and medicine

In this webinar, Adam Ameur of SciLifeLab at Uppsala University shares how he uses Single Molecule, Real-Time (SMRT) Sequencing applications for medical diagnostics and human genetics research, including sequencing of single genes and de novo assembly of human genomes as well as a new method for detection of CRISPR-Cas9 off-targets.

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Tuesday, December 22, 2020

Amplification-free protocol for targeted enrichment of repeat expansion genomic regions and SMRT Sequencing

Many genetic disorders are associated with repeat sequence expansions. Obtaining accurate DNA sequence information from these regions will facilitate researchers to further establish the relationship between these genetic disorders and underlying disease mechanisms. Moreover, repeat interruptions have also been shown to act as phenotypic modifiers in some disorders. Targeted sequencing is an economical way to obtain sequence information from one or more defined regions in a genome. However, most targeted enrichment and sequencing methods require some form of DNA amplification. Amplifying large regions with extreme GC content as seen in repeat expansion disorders is challenging and prone to introducing sequence…

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Tuesday, December 22, 2020

Amplification-free targeted enrichment powered by CRISPR-Cas9 and long-read Single Molecule Real-Time (SMRT) Sequencing can efficiently and accurately sequence challenging repeat expansion disorders

Genomic regions with extreme base composition bias and repetitive sequences have long proven challenging for targeted enrichment methods, as they rely upon some form of amplification. Similarly, most DNA sequencing technologies struggle to faithfully sequence regions of low complexity. This has been especially trying for repeat expansion disorders such as Fragile-X disease, Huntington disease and various Ataxias, where the repetitive elements range from several hundreds of bases to tens of kilobases. We have developed a robust, amplification-free targeted enrichment technique, called No-Amp Targeted Sequencing, that employs the CRISPR-Cas9 system. In conjunction with SMRT Sequencing, which delivers long reads spanning the…

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