scholarly journals In vivo optical imaging-guided targeted sampling for precise diagnosis and molecular pathology

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aditi Sahu ◽  
Yuna Oh ◽  
Gary Peterson ◽  
Miguel Cordova ◽  
Cristian Navarrete-Dechent ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Pathology ◽  
Ex Vivo ◽  
Molecular Profiling ◽  
Next Generation ◽  
Spatial Targeting ◽  
Mutational Profiling ◽  
Generation Sequencing

AbstractConventional tissue sampling can lead to misdiagnoses and repeated biopsies. Additionally, tissue processed for histopathology suffers from poor nucleic acid quality and/or quantity for downstream molecular profiling. Targeted micro-sampling of tissue can ensure accurate diagnosis and molecular profiling in the presence of spatial heterogeneity, especially in tumors, and facilitate acquisition of fresh tissue for molecular analysis. In this study, we explored the feasibility of performing 1–2 mm precision biopsies guided by high-resolution reflectance confocal microscopy (RCM) and optical coherence tomography (OCT), and reflective metallic grids for accurate spatial targeting. Accurate sampling was confirmed with either histopathology or molecular profiling through next generation sequencing (NGS) in 9 skin cancers in 7 patients. Imaging-guided 1–2 mm biopsies enabled spatial targeting for in vivo diagnosis, feature correlation and depth assessment, which were confirmed with histopathology. In vivo 1-mm targeted biopsies achieved adequate quantity and high quality of DNA for next-generation sequencing. Subsequent mutational profiling was confirmed on 1 melanoma in situ and 2 invasive melanomas, using a 505-gene mutational panel called Memorial Sloan Kettering-Integrated mutational profiling of actionable cancer targets (MSK-IMPACT). Differential mutational landscapes, in terms of number and types of mutations, were found between invasive and in situ melanomas in a single patient. Our findings demonstrate feasibility of accurate sampling of regions of interest for downstream histopathological diagnoses and molecular pathology in both in vivo and ex vivo settings with broad diagnostic, therapeutic and research potential in cutaneous diseases accessible by RCM-OCT imaging.

scholarly journals Rapid whole-genome mutational profiling using next-generation sequencing technologies

2008 ◽  
Vol 18 (10) ◽  
pp. 1638-1642 ◽  
Author(s):  
D. R. Smith ◽  
A. R. Quinlan ◽  
H. E. Peckham ◽  
K. Makowsky ◽  
W. Tao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Mutational Profiling ◽  
Generation Sequencing

scholarly journals Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 92 ◽  
Author(s):  
Shannon J. McKie ◽  
Anthony Maxwell ◽  
Keir C. Neuman
Keyword(s):  
Next Generation Sequencing ◽  
Dna Cleavage ◽  
Next Generation ◽  
Dna Breaks ◽  
Genome Wide ◽  
Extension Activity ◽  
Nucleotide Resolution ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

scholarly journals Clinical mutational profiling and categorization of BRAF mutations in melanomas using next generation sequencing

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Parvez M. Lokhandwala ◽  
Li-Hui Tseng ◽  
Erika Rodriguez ◽  
Gang Zheng ◽  
Aparna Pallavajjalla ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Braf Mutations ◽  
Mutational Profiling ◽  
Generation Sequencing

scholarly journals Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Marilia Rita Pinzone ◽  
Maria Paola Bertuccio ◽  
D. Jake VanBelzen ◽  
Ryan Zurakowski ◽  
Una O’Doherty
Keyword(s):  
T Cells ◽  
Next Generation Sequencing ◽  
Cd4 T Cells ◽  
Next Generation ◽  
Activated T Cells ◽  
Selection Pressures ◽  
Hiv Dna ◽  
Generation Sequencing

ABSTRACT Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo. When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

Novel and established EWSR1 gene fusions and associations identified by next-generation sequencing and fluorescence in-situ hybridization

2019 ◽  
Vol 93 ◽  
pp. 65-73 ◽  
Author(s):  
Melissa Krystel-Whittemore ◽  
Martin S. Taylor ◽  
Miguel Rivera ◽  
Jochen K. Lennerz ◽  
Long P. Le ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Next Generation Sequencing ◽  
Fluorescence In Situ Hybridization ◽  
Gene Fusions ◽  
Next Generation ◽  
Ewsr1 Gene ◽  
Generation Sequencing
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