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Assessing the Resolution of Methyltransferase-Mediated DNA Optical Mapping.

ABSTRACT: Interest in the human microbiome is growing and has been, for the past decade, leading to new insights into disease etiology and general human biology. Stimulated by these advances and in a parallel trend, new DNA sequencing platforms have been developed, radically expanding the possibilities in microbiome research. While DNA sequencing plays a pivotal role in this field, there are some technological hurdles that are yet to be overcome. Targeting of the 16S rRNA gene with amplicon sequencing, for instance, is frequently used for sample composition profiling due to its short sample-to-result time and low cost, which counterbalance its low resolution (genus to species level). On the other hand, more comprehensive methods, namely, whole-genome sequencing (WGS) and shallow shotgun sequencing, are capable of yielding single-gene- and functional-level resolution at a higher cost and much higher sample processing time. It goes without saying that the existing gap between these two types of approaches still calls for the development of a fast, robust, and low-cost analytical platform. In search of the latter, we investigated the taxonomic resolution of methyltransferase-mediated DNA optical mapping and found that strain-level identification can be achieved with both global and whole-genome analyses as well as using a unique identifier (UI) database. In addition, we demonstrated that UI selection in DNA optical mapping, unlike variable region selection in 16S amplicon sequencing, is not limited to any genomic location, explaining the increase in resolution. This latter aspect was highlighted by SCCmec typing in methicillin-resistant Staphylococcus aureus (MRSA) using a simulated data set. In conclusion, we propose DNA optical mapping as a method that has the potential to be highly complementary to current sequencing platforms.

SUBMITTER: D'Huys L

PROVIDER: S-EPMC8387989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

Project description:AimsCardiac optical mapping is the gold standard for measuring complex electrophysiology in ex vivo heart preparations. However, new methods for optical mapping in vivo have been elusive. We aimed at developing and validating an experimental method for performing in vivo cardiac optical mapping in pig models.Methods and resultsFirst, we characterized ex vivo the excitation-ratiometric properties during pacing and ventricular fibrillation (VF) of two near-infrared voltage-sensitive dyes (di-4-ANBDQBS/di-4-ANEQ(F)PTEA) optimized for imaging blood-perfused tissue (n = 7). Then, optical-fibre recordings in Langendorff-perfused hearts demonstrated that ratiometry permits the recording of optical action potentials (APs) with minimal motion artefacts during contraction (n = 7). Ratiometric optical mapping ex vivo also showed that optical AP duration (APD) and conduction velocity (CV) measurements can be accurately obtained to test drug effects. Secondly, we developed a percutaneous dye-loading protocol in vivo to perform high-resolution ratiometric optical mapping of VF dynamics (motion minimal) using a high-speed camera system positioned above the epicardial surface of the exposed heart (n = 11). During pacing (motion substantial) we recorded ratiometric optical signals and activation via a 2D fibre array in contact with the epicardial surface (n = 7). Optical APs in vivo under general anaesthesia showed significantly faster CV [120 (63-138) cm/s vs. 51 (41-64) cm/s; P = 0.032] and a statistical trend to longer APD90 [242 (217-254) ms vs. 192 (182-233) ms; P = 0.095] compared with ex vivo measurements in the contracting heart. The average rate of signal-to-noise ratio (SNR) decay of di-4-ANEQ(F)PTEA in vivo was 0.0671 ± 0.0090 min-1. However, reloading with di-4-ANEQ(F)PTEA fully recovered the initial SNR. Finally, toxicity studies (n = 12) showed that coronary dye injection did not generate systemic nor cardiac damage, although di-4-ANBDQBS injection induced transient hypotension, which was not observed with di-4-ANEQ(F)PTEA.ConclusionsIn vivo optical mapping using voltage ratiometry of near-infrared dyes enables high-resolution cardiac electrophysiology in translational pig models.

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Assessing the Resolution of Methyltransferase-Mediated DNA Optical Mapping.

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