October 29, 2021  |  

Targeting Clinically Significant Dark Regions of the Human Genome with High-Accuracy, Long-Read Sequencing

There are many clinically important genes in “dark” regions of the human genome. These regions are characterized as dark due to a paucity of NGS coverage as a result of short-read sequencing or mapping difficulties. Low NGS sequencing yield can arise in these regions due to the presence of various repeat elements or biased base composition while inaccurate mapping can result from segmental duplications. Long-read sequencing coupled with an optimized, robust enrichment method has the potential to illuminate these dark regions. 


October 29, 2021  |  

Resolving Complex Pathogenic Alleles using HiFi Long-Range Amplicon Data and a New Clustering Algorithm

Many genetic diseases are mapped to structurally complex loci. These regions contain highly similar paralogous alleles (>99% identity) that span kilobases within the human genome. Comprehensive screening for pathogenic variants is incomplete and labor intensive using short-reads or optical mapping. In contrast, long-range amplification and PacBio HiFi sequencing fully and directly resolve and phase a wide range of pathogenic variants without inference. To capitalize on the accuracy of HiFi data we designed a new amplicon analysis tool, pbAA. pbAA can rapidly deconvolve a mixture of haplotypes, enabling precise diplotyping, and disease allele classification. 


September 7, 2021  |  

Full-Length Sequencing of CYP2D6 Locus with HiFi Reads Increasing Genotypes Accuracy 

The highly polymorphic CYP2D6 gene impacts the metabolism of 25% of the mostly prescribed drugs. Thus, accurate identification of variant CYP2D6 alleles in individuals is necessary for personalized medicine. PacBio HiFi sequencing produces long and accurate reads to identify variant regions. Here, we describe an end-to-end workflow for the characterization of full-length CYP2D6 by HiFi sequencing. 


September 7, 2021  |  

Targeting Clinically Significant Dark Regions of the Human Genome with High-Accuracy, Long-Read Sequencing

There are many clinically important genes in “dark” regions of the human genome. These regions are characterized as dark due to a paucity of NGS coverage as a result of short-read sequencing or mapping difficulties. Low NGS sequencing yield can arise in these regions due to the presence of various repeat elements or biased base composition while inaccurate mapping can result from segmental duplications. Long-read sequencing coupled with an optimized, robust enrichment method has the potential to illuminate these dark regions. 


September 7, 2021  |  

Resolving Complex Pathogenic Alleles using HiFi Long-Range Amplicon Data and a New Clustering Algorithm

Many genetic diseases are mapped to structurally complex loci. These regions contain highly similar paralogous alleles (>99% identity) that span kilobases within the human genome. Comprehensive screening for pathogenic variants is incomplete and labor intensive using short-reads or optical mapping. In contrast, long-range amplification and PacBio HiFi sequencing fully and directly resolve and phase a wide range of pathogenic variants without inference. To capitalize on the accuracy of HiFi data we designed a new amplicon analysis tool, pbAA. pbAA can rapidly deconvolve a mixture of haplotypes, enabling precise diplotyping, and disease allele classification. 


June 1, 2021  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing in unlocking the underlying biological disease mechanisms of repeat expansion disorders

For many of the repeat expansion disorders, the disease gene has been discovered, however the underlying biological mechanisms have not yet been fully understood. This is mainly due to technological limitations that do not allow for the needed base-pair resolution of the long, repetitive genomic regions. We have developed a novel, amplification-free enrichment technique that uses the CRISPR/Cas9 system to target large repeat expansions. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of these complex genomic regions. By using a PCR-free amplification method, we are able to access not only the repetitive elements and interruption sequences accurately, but also the epigenetic information.


June 1, 2021  |  

Candidate gene screening using long-read sequencing

We have developed several candidate gene screening applications for both Neuromuscular and Neurological disorders. The power behind these applications comes from the use of long-read sequencing. It allows us to access previously unresolvable and even unsequencable genomic regions. SMRT Sequencing offers uniform coverage, a lack of sequence context bias, and very high accuracy. In addition, it is also possible to directly detect epigenetic signatures and characterize full-length gene transcripts through assembly-free isoform sequencing. In addition to calling the bases, SMRT Sequencing uses the kinetic information from each nucleotide to distinguish between modified and native bases.


June 1, 2021  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders

Nucleotide repeat expansions are a major cause of neurological and neuromuscular disease in humans, however, the nature of these genomic regions makes characterizing them extremely challenging. Accurate DNA sequencing of repeat expansions using short-read sequencing technologies is difficult, as short-read technologies often cannot read through regions of low sequence complexity. Additionally, these short reads do not span the entire region of interest and therefore sequence assembly is required. Lastly, most target enrichment methods are reliant upon amplification which adds the additional caveat of PCR bias. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific targeting of individual human genes. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples and this strategy, we have successfully targeted the loci of Huntington’s Disease (HTT; CAG repeat), Fragile X (FMR1; CGG repeat), ALS (C9orf72; GGGGCC repeat), and Spinocerebellar ataxia type 10 (SCA10; variable ATTCT repeat) for examination. With this data, we demonstrate the ability to isolate hundreds of individual on-target molecules in a single SMRT Cell and accurately sequence through long repeat stretches, regardless of the extreme GC-content. The method is compatible with multiplexing of multiple targets and multiple samples in a single reaction. This technique also captures native DNA molecules for sequencing, allowing for the possibility of direct detection and characterization of epigenetic signatures.


June 1, 2021  |  

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 novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific targeting of individual human genes. This method, in conjunction with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows the sequencing of complex genomic regions that cannot be investigated with other technologies.


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