Next-generation sequencing (NGS) technologies using DNA, RNA, or methylation se‐ quencing have impacted enormously on the life sciences. NGS is the choice for large-scale genomic and transcriptomic sequencing because of the high-throughput production and outputs of sequencing data in the gigabase range per instrument run and the lower cost compared to the traditional Sanger first-generation sequencing method. The vast amounts of data generated by NGS have broadened our understanding of structural and functional genomics through the concepts of “omics” ranging from basic genomics to in‐ tegrated systeomics, providing new insight into the workings and meaning of genetic conservation and diversity of living things. NGS today is more than ever about how dif‐ ferent organisms use genetic information and molecular biology to survive and repro‐ duce with and without mutations, disease, and diversity within their population networks and changing environments. In this chapter, the advances, applications, and challenges of NGS are reviewed starting with a history of first-generation sequencing fol‐ lowed by the major NGS platforms, the bioinformatics issues confronting NGS data stor‐ age and analysis, and the impacts made in the fields of genetics, biology, agriculture, and medicine in the brave, new world of ”omics.”

/pdf/next-generation-sequencing-an-overview-of-the-history-tools-1etds00fra.pdf

Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications

Efficient and cost-effective DNA sequencing technologies are critical to the progress of molecular biology. This overview of DNA sequencing strategies provides a high-level review of seven distinct approaches to DNA sequencing: (a) dideoxy sequencing; (b) solid phase sequencing; (c) sequencing-by-hybridization; (d) mass spectrometry; (e) cyclic array sequencing; (f) microelectrophoresis; and (g) nanopore sequencing. Other platforms currently in development are also briefly described. The primary focus here is on Sanger dideoxy sequencing, which has been the dominant technology since 1977, and on cyclic array strategies, for which several competitive implementations have been developed since 2005. Because the field of DNA sequencing is changing rapidly, this unit represents a snapshot as of September, 2011.

https://arep.med.harvard.edu/pdf/Shendure08a.pdf

Overview of DNA sequencing strategies.

Direct imaging becomes important when the knowledge at few/single molecule level is requested and where the diffraction does not allow to get structural and functional information. Here we report on the direct imaging of double stranded (ds) λ-DNA in the A conformation, obtained by combining a novel sample preparation method based on super hydrophobic DNA molecules self-aggregation process with transmission electron microscopy (TEM). The experimental breakthrough is the production of robust and highly ordered paired DNA nanofibers that allowed its direct TEM imaging and the double helix structure revealing.

/pdf/direct-imaging-of-dna-fibers-the-visage-of-double-helix-2taku4et8p.pdf

Direct imaging of DNA fibers: the visage of double helix.

Next-generation sequencing for constitutional variants in the clinical laboratory, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

The correction of electron lens aberrations

Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/CC8E9C69093282A80BF02823F1AEEC0B/S1431927612012615a.pdf/div-class-title-dna-base-identification-by-electron-microscopy-div.pdf

DNA base identification by electron microscopy.

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5′ and 3′ termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.

/pdf/a-novel-crispr-cas9-associated-technology-for-sequence-1yqc2jdlor.pdf

A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.

Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.

Detection of targeted sequence regions

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

/pdf/albert-crewe-s-dream-realized-sequencing-dna-with-stem-mqt3cs4ias.pdf

Albert Crewe's Dream Realized: Sequencing DNA with STEM

/pdf/dna-sequencing-with-tem-55h67e950g.pdf

DNA Sequencing with TEM

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