Abstract:Analysis of structural variations
(SVs) is important to understand
mutations underlying genetic disorders and pathogenic conditions.
However, characterizing SVs using short-read, high-throughput sequencing
technology is difficult. Although long-read sequencing technologies
are being increasingly employed in characterizing SVs, their low throughput
and high costs discourage widespread adoption. Sequence motif-based
optical mapping in nanochannels is useful in whole-genome mapping
and SV detection, but it is not… Show more
“…However, without PCR enrichment, one would need more genomic input DNA and more amount of data to achieve similar coverages at target loci, adding to cost. For more complex SVs, multi-color whole-genome mapping can be used to create custom maps of the sample and validate the combinations of gRNA to be used in an experiment 20 , 29 , 30 . Using our universal approach, it is possible to target multiple SVs in a sample or analyze an SV across multiple samples, enabling precise detection of breakpoints economically.…”
Section: Discussionmentioning
confidence: 99%
“…Several reports have been published where optical mapping was used to detect SVs (1 kbp—several Mbp) including some complex cases 11 , 15 , 20 , 21 . Although the precise location of the SV breakpoints and sequence information is lacking, the SV region obtained from optical mapping can be leveraged subsequently for targeted sequencing.…”
Identification of structural variants (SVs) breakpoints is important in studying mutations, mutagenic causes, and functional impacts. Next-generation sequencing and whole-genome optical mapping are extensively used in SV discovery and characterization. However, multiple platforms and computational approaches are needed for comprehensive analysis, making it resource-intensive and expensive. Here, we propose a strategy combining optical mapping and cas9-assisted targeted nanopore sequencing to analyze SVs. Optical mapping can economically and quickly detect SVs across a whole genome but does not provide sequence-level information or precisely resolve breakpoints. Furthermore, since only a subset of all SVs is known to affect biology, we attempted to type a subset of all SVs using targeted nanopore sequencing. Using our approach, we resolved the breakpoints of five deletions, five insertions, and an inversion, in a single experiment.
“…However, without PCR enrichment, one would need more genomic input DNA and more amount of data to achieve similar coverages at target loci, adding to cost. For more complex SVs, multi-color whole-genome mapping can be used to create custom maps of the sample and validate the combinations of gRNA to be used in an experiment 20 , 29 , 30 . Using our universal approach, it is possible to target multiple SVs in a sample or analyze an SV across multiple samples, enabling precise detection of breakpoints economically.…”
Section: Discussionmentioning
confidence: 99%
“…Several reports have been published where optical mapping was used to detect SVs (1 kbp—several Mbp) including some complex cases 11 , 15 , 20 , 21 . Although the precise location of the SV breakpoints and sequence information is lacking, the SV region obtained from optical mapping can be leveraged subsequently for targeted sequencing.…”
Identification of structural variants (SVs) breakpoints is important in studying mutations, mutagenic causes, and functional impacts. Next-generation sequencing and whole-genome optical mapping are extensively used in SV discovery and characterization. However, multiple platforms and computational approaches are needed for comprehensive analysis, making it resource-intensive and expensive. Here, we propose a strategy combining optical mapping and cas9-assisted targeted nanopore sequencing to analyze SVs. Optical mapping can economically and quickly detect SVs across a whole genome but does not provide sequence-level information or precisely resolve breakpoints. Furthermore, since only a subset of all SVs is known to affect biology, we attempted to type a subset of all SVs using targeted nanopore sequencing. Using our approach, we resolved the breakpoints of five deletions, five insertions, and an inversion, in a single experiment.
“…Cas9-mediated nick labeling can be combined with traditional enzymatic nick labeling, to create multicolor optical maps and enable both global sequence motif mapping and labeling directed specifically to a sequence of interest, which has found applications in structural variation analysis and telomere characterization (McCaffrey et al, 2016(McCaffrey et al, , 2017Levy-Sakin et al, 2019;. Uppuluri et al (2021) recently presented another multicolor labeling approach by combining Cas9-mediated nick labeling and the enzymatic direct labeling technology from BioNano Genomics (see section 'Recent applications of optical DNA mapping'). They validated the usefulness of their methodology by quantifying copy numbers of D4Z4 repeats on chromosome 4q, detecting long non-interspersed elements 1 (LINE-1) insertions, and estimating telomere lengths.…”
Section: Enzyme-based Labelingmentioning
confidence: 99%
“…The contour of the DNA is visualized by staining the backbone of the DNA molecule, typically with the bis-intercalating dye YOYO-1. Several additions have been made to expand the nick labeling toolbox, including nick-flap labeling (Das et al ., 2010), dual nick labeling (Hastie et al ., 2013), and highly specific Cas9-based labeling strategies (McCaffrey et al ., 2016; Zhang et al ., 2018; Abid et al ., 2020; Uppuluri et al ., 2021). Fig.…”
Section: Optical Dna Mappingmentioning
confidence: 99%
“…Uppuluri et al . (2021) recently presented another multicolor labeling approach by combining Cas9-mediated nick labeling and the enzymatic direct labeling technology from BioNano Genomics (see section ‘Recent applications of optical DNA mapping’). They validated the usefulness of their methodology by quantifying copy numbers of D4Z4 repeats on chromosome 4q, detecting long non-interspersed elements 1 (LINE-1) insertions, and estimating telomere lengths.…”
Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinitybased labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.
Purpose of Review
The length of telomeres, protective structures at the chromosome ends, is a well-established biomarker for pathological conditions including multisystemic syndromes called telomere biology disorders. Approaches to measure telomere length (TL) differ on whether they estimate average, distribution, or chromosome-specific TL, and each presents their own advantages and limitations.
Recent Findings
The development of long-read sequencing and publication of the telomere-to-telomere human genome reference has allowed for scalable and high-resolution TL estimation in pre-existing sequencing datasets but is still impractical as a dedicated TL test. As sequencing costs continue to fall and strategies for selectively enriching telomere regions prior to sequencing improve, these approaches may become a promising alternative to classic methods.
Summary
Measurement methods rely on probe hybridization, qPCR or more recently, computational methods using sequencing data. Refinements of existing techniques and new approaches have been recently developed but a test that is accurate, simple, and scalable is still lacking.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
