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Super-Resolution Genome Mapping in Silicon Nanochannels

Jeffet, Jonathan; Kobo, Asaf; Su, Tianxiang; Grunwald, Assaf; Green, Ori; Kinos, Adam LU ; Eisenberg, Eli; Ambjörnsson, Tobias LU ; Westerlund, Fredrik and Weinhold, Elmar, et al. (2016) In ACS Nano 10(11). p.9823-9830
Abstract

Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping... (More)

Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value scores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition.

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publication status
published
subject
keywords
confined polymers, DNA labeling, nanochannels, optical genome mapping, single-molecule, super-resolution
in
ACS Nano
volume
10
issue
11
pages
8 pages
publisher
The American Chemical Society
external identifiers
  • scopus:84997471114
  • wos:000388913100010
ISSN
1936-0851
DOI
10.1021/acsnano.6b05398
language
English
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yes
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c2027e95-4e42-4177-be24-530e29e83b97
date added to LUP
2016-12-12 08:33:39
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2017-11-12 04:27:11
@article{c2027e95-4e42-4177-be24-530e29e83b97,
  abstract     = {<p>Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value scores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition.</p>},
  author       = {Jeffet, Jonathan and Kobo, Asaf and Su, Tianxiang and Grunwald, Assaf and Green, Ori and Kinos, Adam and Eisenberg, Eli and Ambjörnsson, Tobias and Westerlund, Fredrik and Weinhold, Elmar and Shabat, Doron and Purohit, Prashant K. and Ebenstein, Yuval},
  issn         = {1936-0851},
  keyword      = {confined polymers,DNA labeling,nanochannels,optical genome mapping,single-molecule,super-resolution},
  language     = {eng},
  month        = {11},
  number       = {11},
  pages        = {9823--9830},
  publisher    = {The American Chemical Society},
  series       = {ACS Nano},
  title        = {Super-Resolution Genome Mapping in Silicon Nanochannels},
  url          = {http://dx.doi.org/10.1021/acsnano.6b05398},
  volume       = {10},
  year         = {2016},
}