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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.


June 1, 2021  |  

Targeted enrichment without amplification 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 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. Using human genomic DNA samples and this strategy, we have successfully targeted the loci of a number of repeat expansion disorders (HTT, FMR1, ATXN10, C9orf72). With this data, we demonstrate the ability to isolate hundreds of individual on-target molecules and accurately sequence through long repeat stretches, regardless of the extreme GC-content, followed by accurate sequencing on a single PacBio RS II SMRT Cell or Sequel SMRT Cell 1M. The method is compatible with multiplexing of multiple targets and multiple samples in a single reaction. Furthermore, this technique also preserves native DNA molecules for sequencing, allowing for the possibility of direct detection and characterization of epigenetic signatures. We demonstrate detection of 5-mC in human promoter sequences and CpG islands.


June 1, 2021  |  

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion 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 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.


June 1, 2021  |  

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

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.


April 21, 2020  |  

A Novel Bacteriophage Exclusion (BREX) System Encoded by the pglX Gene in Lactobacillus casei Zhang.

The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (?pglX) was constructed. Interestingly, m6A methylation of the 5′-ACRCAG-3′ motif was eliminated in the ?pglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ?pglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5′-ACRCAG-3′). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ?pglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria. Copyright © 2019 American Society for Microbiology.


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