Super-Resolution Genome Mapping in Silicon Nanochannels

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.

Analytical epigenetics: single-molecule optical detection of DNA and histone modifications

“…After tagging with fluorophores, the unmethylated sites (or, the ‘unmethylome’) can be read out by genome-wide optical methylation mapping [38] (Figure 3 (b)), allowing the analysis of large structural aberrations such as pathogenic macrosatellite arrays. Future implementation of super-resolution mapping promises to locate the epigenetic modifications more precisely, possibly resolving the detailed methylation status of CpG islands [39]. The lack of direct covalent labeling strategies for 5mC can also be bypassed by transforming 5mC into 5hmC with Tet enzymes [40].…”

Reduced representation optical methylation mapping (R2OM2)

“…We used red fluorophores for the genetic barcode and green fluorophores for methylation mapping. On the non-methylated BAC, M.TaqI creates a unique pattern along the non-repetitive region and has two close-by recognition sites on each repeat unit (resulting in one visual label on each repeat, due to diffraction limits) 49 (Figure 5a). As expected, the non-methylated BAC DNA was dually labeled by both enzymes and thus contained two barcode layers, in agreement with the theoretical dual-color barcode ( Figure 5b).…”

Die Methyltransferase‐gesteuerte Markierung von Biomolekülen und ihre Anwendungen

“…Die Schwellenwertbildung der Kreuzkorrelationsergebnisse ermöglichte den effizienten Abgleich der Bakteriophagen mit der korrekten Sequenz. Erst kürzlich demonstrierte dieselbe Gruppe außerdem, dass die Genom‐Kartierung mit einer Auflösung unterhalb der Beugungsgrenze in Silicium‐Nanokanälen durchgeführt werden kann, wodurch die Informationsdichte deutlich erhöht wird …”