Extension distribution for DNA confined in a nanochannel near the Odijk regime

Abstract

DNA confinement in a nanochannel typically is understood via mapping to the confinement of an equivalent neutral polymer by hard walls. This model has proven to be effective for confinement in relatively large channels where hairpin formation is frequent. An analysis of existing experimental data for Escherichia coli DNA extension in channels smaller than the persistence length, combined with an additional dataset for λ-DNA confined in a 34 nm wide channel, reveals a breakdown in this approach as the channel size approaches the Odijk regime of strong confinement. In particular, the predicted extension distribution obtained from the asymptotic solution to the weakly correlated telegraph model for a confined wormlike chain deviates significantly from the experimental distribution obtained for DNA confinement in the 34 nm channel, and the discrepancy cannot be resolved by treating the alignment fluctuations or the effective channel size as fitting parameters. We posit that the DNA-wall electrostatic interactions, which are sensible throughout a significant fraction of the channel cross section in the Odijk regime, are the source of the disagreement between theory and experiment. Dimensional analysis of the wormlike chain propagator in channel confinement reveals the importance of a dimensionless parameter, reflecting the magnitude of the DNA-wall electrostatic interactions relative to thermal energy, which has not been considered explicitly in the prevailing theories for DNA confinement in a nanochannel.

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

“…As shown and calculated in some recent works [ 17 , 45–48 ], during the exposure time of a single frame (∼150–200 ms in the commercial system) the thermal fluctuations smear the markers' signal, which limits the resolution of two adjacent markers to ∼1.5–3 kbp separation. This value is larger than the optical diffraction limit discussed above and therefore the fluctuations are the main limit on the achievable resolution.…”

“…However, optical mapping is generally done in nanochannels that have cross-sections on the same scale as the DNA persistence length, and therefore deviates from this ideal regime [45,47,48]. Adjustments to the Odijk theory have been extensively studied in recent years, both theoretically [17,46,[50][51][52][53][54] and experimentally [17,45,47,48,[55][56][57][58], and we refer the readers to two excellent reviews on the subject [6,59]. The key concepts relevant here are that deviations from the fully extended classic Odijk regime can be manifested as backfolding, hairpins, loops and bunching of the DNA molecules inside the channels (leftmost panel in Figure 3A.).…”

“…The key concepts relevant here are that deviations from the fully extended classic Odijk regime can be manifested as backfolding, hairpins, loops and bunching of the DNA molecules inside the channels (leftmost panel in Figure 3 A.). These deviations were incorporated into recent theoretical models [ 51 , 60 ], albeit minor discrepancies still remain between theory and experimental data of molecules confined to <50 × 50 nm² cross-sections channels [ 17 , 61 , 62 ]. As a result of this research, an important reduction of nanochannels’ widths from 45 nm to 34 nm, was introduced by BioNano Genomics in their commercial Saphyr chips for optical genome mapping.…”

“…As a result of this research, an important reduction of nanochannels’ widths from 45 nm to 34 nm, was introduced by BioNano Genomics in their commercial Saphyr chips for optical genome mapping. This allows better DNA extension and therefore increased genomic resolution, and reduced alignment errors due to thermal fluctuations [ 17 , 45 ]. Despite remaining limitations [ 62 ], the current framework is well-adequate to allow the extraction of valuable genetic and epigenetic information as will be discussed in the following sections.…”

“…Optical mapping is commonly compared against other sequencing methods that offer long reads with single-molecule sensitivity, such as Oxford Nanopore [ 14 ] or Pacific Biosciences’ (PacBio) single-molecule real-time (SMRT) sequencing [ 15 ]. Unlike optical mapping, which is limited in its resolution (>500 bp for ensemble averaged SV detection [ 16 ], and optical resolution of ∼1500–2500 bp [ 17 ]), these methods offer single base-pair resolution with longer reads compared to NGS. Although these emerging technologies are pushing the limits of sequencing and can already capture medium-scale SVs with high precision [ 18 ], they are still limited in the fraction of ultra-long reads and price per genome coverage (see Table 1 ).…”