Optical mapping of DNA: Single‐molecule‐based methods for mapping genomes

Abstract

The technologies associated with DNA sequencing are rapidly evolving. Indeed, single-molecule DNA sequencing strategies are cheaper and faster than ever before. Despite this progress, every sequencing platform to date relies on reading the genome in small, abstract fragments, typically of less than 1000 bases in length. The overarching aim of the optical map is to complement the information derived from DNA sequencing by providing long-range context on which these short sequence reads can be built. This is typically done using an enzyme to target and modify at short DNA sequences of, say, six bases in length throughout the genome. By accurately placing these short pieces of sequence on long genomic DNA fragments, up to several millions of bases in length, a scaffold for sequence assembly can be obtained. This review focuses on three enzymatic approaches to optical mapping. Optical mapping was first developed using restriction enzymes to sequence-specifically cleave DNA that is immobilized on a surface. More recently, nicking enzymes have found application in the sequence-specific fluorescent labeling of DNA for optical mapping. Such covalent modification allows the DNA to be imaged in solution, and this, in combination with developing nanofluidic technologies, is enabling new high-throughput approaches to mapping. And, finally, this review will discuss the recent development of mapping with subdiffraction-limit precision using methyltransferase enzymes to label the DNA with an ultrahigh density.

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…This gave a high density of modified sites, which were subsequently labelled using NHS ester or azide derivatives of the Atto647N dye. Following deposition, sub‐diffraction‐limit imaging was achieved based on stochastic photobleaching and localization of individual emitters 76a. This resulted in an approximate resolution of 42 nm (approximately 80 base pairs) 19c…”

“…One example is the typing of bacteriophage DNA molecules based on a barcode embedded in the molecule. Another application could be the detection of copy‐number variation, repeats of large genomic elements in the genomes that are hard to find by sequencing 76a. Finally, the MTase‐based labeling approach can easily be combined with other genomic analysis approaches such as fluorescence in situ hybridization (FISH).…”

High Throughput Sequencing Advances and Future Challenges

“…Rationalized techniques to assemble high-quality whole genome sequences were needed, particularly in the microbial genomics of Gram-negative multidrug-resistant, somewhere de novo sequencing technique is constantly mandatory because of the genetic multiplicity and vibrant genome reorganization take place [43,44]. WGM utilize singlemolecule restriction examination to acquire results concerning the magnitude of the control splinter and their substantial positions beside the DNA strand [45]. Whole genome has been exploit in a number of functions, which comprised of phylogenetic analyses and genotyping of associated with microbial isolates [46,47], discovery of outsized genomic structural rearrangements or variations [48,49], and quality control or verification for genome sequences which were assembled [50,51].…”

Automation of a single-DNA molecule stretching device

“…Microfluidics has emerged as a key technology that enables extraction, amplification, and detection of nucleic acids in lab-on-a-chip format, 1 which is relevant for a range of applications such as pathogen detection or whole genome analysis. In particular, the DNA sequence analysis via fluorescence imaging of stretched genomic-length DNA has emerged as a method complementary to DNA sequencing 2 with the perspective of analysing single DNA molecules.…”