Super-Resolution Genome Mapping in Silicon Nanochannels

Research output: Contribution to journalArticle

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 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.

Details

Authors
  • Jonathan Jeffet
  • Asaf Kobo
  • Tianxiang Su
  • Assaf Grunwald
  • Ori Green
  • Adam Kinos
  • Eli Eisenberg
  • Tobias Ambjörnsson
  • Fredrik Westerlund
  • Elmar Weinhold
  • Doron Shabat
  • Prashant K. Purohit
  • Yuval Ebenstein
Organisations
External organisations
  • Harvard University
  • Tel-Aviv University
  • Chalmers University of Technology
  • University of Pennsylvania
  • RWTH Aachen University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Nano Technology
  • Genetics

Keywords

  • confined polymers, DNA labeling, nanochannels, optical genome mapping, single-molecule, super-resolution
Original languageEnglish
Pages (from-to)9823-9830
Number of pages8
JournalACS Nano
Volume10
Issue number11
Publication statusPublished - 2016 Nov 22
Publication categoryResearch
Peer-reviewedYes