ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization

Hu, Mengwei; Yang, Bing; Cheng, Yubao; Radda, Jonathan S. D.; Chen, Yanbo; Liu, Miao; Wang, Siyuan

doi:10.1038/s41598-020-76439-x

Cited by 31 publications

(27 citation statements)

References 20 publications

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“…Each template oligo contains (from 5′ to 3′): (1) a 20-nt forward priming sequence, (2) a 20-nt secondary probe binding sequence, (3) a 30-nt genome targeting sequence, (4) a same 20-nt secondary probe binding sequence, and (5) a 20-nt reverse priming sequence. The 30-nt genome targeting sequences were designed with ProbeDealer [ 60 ]. The secondary probe binding sequences and priming sequences were introduced in previous studies [ 30 , 43 ].…”

Section: Methodsmentioning

confidence: 99%

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TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

Cheng

Liu

et al. 2021

Genome Biol

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Background Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains. Results Here we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components and transcription mostly do not affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains. Conclusions In sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

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Section: Methodsmentioning

confidence: 99%

“…To design intron RNA FISH probes for genes TMSB4X and RPL36A , we downloaded intron transcript sequences of the genes from UCSC genome browser, and designed template oligo pools for the intron RNA FISH using ProbeDealer [ 60 ]. A total of 24 template oligos were designed for TMSB4X and 45 template oligos were designed for RPL36A .…”

Section: Methodsmentioning

confidence: 99%

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

Cheng

Liu

et al. 2021

Genome Biol

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“…Moreover, the application of Oligopaints is confined to popular model organisms with well-assembled genomes. Another difficulty concerns an extensive bioinformatic expertise to decide on the essential parameters needed to filter the initial oligonucleotide pool, even though multiple tools for designing custom arrays of oligonucleotides for genomic regions have been suggested, including OligoArray ( Rouillard et al, 2003 ), PROBER ( Navin et al, 2006 ), Chorus ( Han et al, 2015 ), OligoMiner ( Beliveau et al, 2018 ), iFISH ( Gelali et al, 2019 ), AnthOligo ( Jayaraman et al, 2020 ), ProbeDealer ( Hu et al, 2020 ). The array-synthesized Oligopaint probes are expensive, compared to BAC- or PCR-based probes for the same-sized genomic regions; however, the cost of probes per hybridization could be comparable if high-throughput FISH is performed (see for discussion Beliveau et al, 2012 ; Boettiger and Murphy, 2020 ).…”

Section: Fish-probes For Chromatin Domain Visualizationmentioning

confidence: 99%

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains

Maslova

Krasikova

2021

Front. Cell Dev. Biol.

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The intimate relationships between genome structure and function direct efforts toward deciphering three-dimensional chromatin organization within the interphase nuclei at different genomic length scales. For decades, major insights into chromatin structure at the level of large-scale euchromatin and heterochromatin compartments, chromosome territories, and subchromosomal regions resulted from the evolution of light microscopy and fluorescence in situ hybridization. Studies of nanoscale nucleosomal chromatin organization benefited from a variety of electron microscopy techniques. Recent breakthroughs in the investigation of mesoscale chromatin structures have emerged from chromatin conformation capture methods (C-methods). Chromatin has been found to form hierarchical domains with high frequency of local interactions from loop domains to topologically associating domains and compartments. During the last decade, advances in super-resolution light microscopy made these levels of chromatin folding amenable for microscopic examination. Here we are reviewing recent developments in FISH-based approaches for detection, quantitative measurements, and validation of contact chromatin domains deduced from C-based data. We specifically focus on the design and application of Oligopaint probes, which marked the latest progress in the imaging of chromatin domains. Vivid examples of chromatin domain FISH-visualization by means of conventional, super-resolution light and electron microscopy in different model organisms are provided.

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“…ProbeDealer is a recently developed tool to design oligo-based FISH probes [59]. It utilizes features such as melting temperature, GC content, secondary structure and dimers to filter oligos detected from genomes based on a sliding window method.…”

Section: Development and Application Of Oligo-fish Probe Design Toolsmentioning

confidence: 99%

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation

Liu

Zhang

2021

IJMS

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Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.

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ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization

Cited by 31 publications

References 20 publications

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation

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