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Thursday, November 12, 2020

Application Brief: No-Amp targeted sequencing – Best Practices

With the PacBio no-amplification (No-Amp) targeted sequencing method, you can now sequence through previously inaccessible regions of the genome to provide base-level resolution of disease-causing repeat expansions. By combining the CRISPR/Cas9 enrichment method with Single Molecule, Real-Time (SMRT) Sequencing on the Sequel Systems you are no longer limited by hard-to-amplify targets.

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Sunday, October 25, 2020

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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Sunday, October 25, 2020

ASHG PacBio Workshop: Going beyond the $1,000 genome? – the future of high quality de novo human genomes, epigenomes and transcriptomes?

Jonas Korlach, Chief Scientific Officer at PacBio, discussed the technology waves that have followed the initial human genome sequencing project, where we are today, and where we are going. Today, we are in what Korlach calls the 4th wave, where more comprehensive whole-genome re-sequencing is occurring, and we are nearing the 5th, when we will actually be able to free ourselves from reference genomes and sequence everything de novo.

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Sunday, October 25, 2020

ASHG Virtual Poster: Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders

PacBio’s Jenny Ekholm presents this ASHG 2016 poster on a new method being developed that enriches for unamplified DNA and uses SMRT Sequencing to characterize repeat expansion disorders. Incorporating the CRISPR/Cas9 system to target specific genes allows for amplification-free enrichment to preserve epigenetic information and avoid PCR bias. Internal studies have shown that the approach can successfully be used to target and sequence the CAG repeat responsible for Huntington’s disease, the repeat associated with ALS, and more. The approach allows for pooling many samples and sequencing with a single SMRT Cell.

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Sunday, October 25, 2020

Webinar: An introduction to PacBio’s long-read sequencing & how it has been used to make important scientific discoveries

In this Webinar, we will give an introduction to Pacific Biosciences’ single molecule, real-time (SMRT) sequencing. After showing how the system works, we will discuss the main features of the technology with an emphasis on the difference between systematic error and random error and how SMRT sequencing produces better consensus accuracy than other systems. Following this, we will discuss several ground-breaking discoveries in medical science that were made possible by the longs reads and high accuracy of SMRT Sequencing.

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Sunday, October 25, 2020

Webinar: Addressing “NGS Dead Zones” with third generation PacBio sequencing

SMRT Sequencing is a DNA sequencing technology characterized by long read lengths and high consensus accuracy, regardless of the sequence complexity or GC content of the DNA sample. These characteristics can be harnessed to address medically relevant genes, mRNA transcripts, and other genomic features that are otherwise difficult or impossible to resolve. I will describe examples for such new clinical research in diverse areas, including full-length gene sequencing with allelic haplotype phasing, gene/pseudogene discrimination, sequencing extreme DNA contexts, high-resolution pharmacogenomics, biomarker discovery, structural variant resolution, full-length mRNA isoform cataloging, and direct methylation detection.

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Sunday, October 25, 2020

AGBT PacBio Workshop: SMRT Sequencing roadmap: better throughput, lower costs

In this AGBT 2017 talk, PacBio CSO Jonas Korlach provided a technology roadmap for the Sequel System, including plans the continue performance and throughput increases through early 2019. Per SMRT Cell throughput of the Sequel System is expected to double this year and again next year. Together with a new higher-capacity SMRT Cell expected to be released by the end of 2018, these improvements result in a ~30-fold increase or ~150 Gb / SMRT Cell allowing a real $1000 real de novo human genome assembly. Also discussed: Additional application protocol improvements, new chemistry and software updates, and a look at…

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Sunday, October 25, 2020

AGBT Virtual Poster: Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method

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 are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. We have developed a…

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Sunday, October 25, 2020

User Group Meeting: Targeted PacBio Sequencing using Sage HLS-CATCH

In this PacBio User Group Meeting presentation, Mount Sinai’s Ethan Ellis presents results from the HLS-CATCH method, which involves the use of the SageHLS instrument with CRISPR design methods to target and extract large genomic fragments for sequencing while avoiding pseudogenes and other confounding regions.

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Sunday, October 25, 2020

AGBT Presentation: Studying CRISPR guide RNA specificity by amplification-free long-read sequencing

At AGBT 2020, Adam Ameur from Uppsala University discussed the use of long-read PacBio sequencing to detect off-target results from CRISPR/Cas9 gene editing studies. His team uses HiFi reads from the Sequel II System to perform whole genome sequencing and figure out exactly where guide RNAs bind. In one example using a human embryonic kidney cell line, they found 55 off-target sites for three guide RNAs. Ameur’s group has already generated preliminary data on results from editing living cells.

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Sunday, October 25, 2020

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, April 21, 2020

Long-read sequencing for rare human genetic diseases.

During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is

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Tuesday, April 21, 2020

Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting…

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Tuesday, April 21, 2020

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~?1.6% in the T2 generation, through PCR-based screening of numerous individuals. However,…

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Tuesday, April 21, 2020

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity…

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