Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Abstract Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

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Refining Noninvasive Prenatal Diagnosis with Single-Molecule Next-Generation Sequencing

Clinical Chemistry ◽

10.1373/clinchem.2011.181388 ◽

2012 ◽

Vol 58 (4) ◽

pp. 657-658 ◽

Cited By ~ 5

Author(s):

Neil D Avent

Keyword(s):

Prenatal Diagnosis ◽

Next Generation Sequencing ◽

Single Molecule ◽

Next Generation ◽

Noninvasive Prenatal Diagnosis ◽

Generation Sequencing

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Identification of novel GNAS mutations in intramuscular myxoma using next-generation sequencing with single-molecule tagged molecular inversion probes

Diagnostic Pathology ◽

10.1186/s13000-019-0787-3 ◽

2019 ◽

Vol 14 (1) ◽

Cited By ~ 1

Author(s):

Elise M. Bekers ◽

Astrid Eijkelenboom ◽

Paul Rombout ◽

Peter van Zwam ◽

Suzanne Mol ◽

...

Keyword(s):

Next Generation Sequencing ◽

Single Molecule ◽

Intramuscular Myxoma ◽

Next Generation ◽

Molecular Inversion Probes ◽

Generation Sequencing

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Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing—A Prospective Feasibility Study

Cancers ◽

10.3390/cancers13123101 ◽

2021 ◽

Vol 13 (12) ◽

pp. 3101

Author(s):

Marina Berger ◽

Andrea Thueringer ◽

Doritt Franz ◽

Nadia Dandachi ◽

Emina Talakić ◽

...

Keyword(s):

Next Generation Sequencing ◽

Metastatic Melanoma ◽

Single Molecule ◽

Progression Free Survival ◽

Circulating Tumor Dna ◽

Next Generation ◽

Dna Amount ◽

Melanoma Patients ◽

Tumor Dna ◽

Generation Sequencing

We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.

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Fragmentation of Surface Adsorbed and Aligned DNA Molecules using Soft Lithography for Next-Generation Sequencing

Journal of Biosensors & Bioelectronics ◽

10.4172/2155-6210.1000247 ◽

2017 ◽

Vol 08 (04) ◽

Cited By ~ 1

Author(s):

NaHyun Cho ◽

Sara Goodwin ◽

Julia Budassi ◽

Ke Zhu ◽

William Richard McCombie ◽

...

Keyword(s):

Next Generation Sequencing ◽

Soft Lithography ◽

Next Generation ◽

Dna Molecules ◽

Generation Sequencing

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Next-Generation Sequencing Technologies and Neurogenetic Diseases

Life ◽

10.3390/life11040361 ◽

2021 ◽

Vol 11 (4) ◽

pp. 361

Author(s):

Hui Sun ◽

Xiao-Rong Shen ◽

Zi-Bing Fang ◽

Zong-Zhi Jiang ◽

Xiao-Jing Wei ◽

...

Keyword(s):

Next Generation Sequencing ◽

Single Molecule ◽

De Novo ◽

Neurological Diseases ◽

Genetic Diseases ◽

Spinocerebellar Ataxias ◽

Next Generation ◽

De Novo Mutations ◽

Coverage Area ◽

Generation Sequencing

Next-generation sequencing (NGS) technology has led to great advances in understanding the causes of Mendelian and complex neurological diseases. Owing to the complexity of genetic diseases, the genetic factors contributing to many rare and common neurological diseases remain poorly understood. Selecting the correct genetic test based on cost-effectiveness, coverage area, and sequencing range can improve diagnosis, treatments, and prevention. Whole-exome sequencing and whole-genome sequencing are suitable methods for finding new mutations, and gene panels are suitable for exploring the roles of specific genes in neurogenetic diseases. Here, we provide an overview of the classifications, applications, advantages, and limitations of NGS in research on neurological diseases. We further provide examples of NGS-based explorations and insights of the genetic causes of neurogenetic diseases, including Charcot–Marie–Tooth disease, spinocerebellar ataxias, epilepsy, and multiple sclerosis. In addition, we focus on issues related to NGS-based analyses, including interpretations of variants of uncertain significance, de novo mutations, congenital genetic diseases with complex phenotypes, and single-molecule real-time approaches.

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