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.

Optical Mapping: Detecting Genomic Resistance Cassettes in MRSA

“…This work constitutes a proof of concept for the use of Fluorocode OM for detection of genomic elements, SCC mec of MRSA in particular, an idea that was previously examined by our group in silico . With previous findings by other optical mapping methods showing the detection of plasmids at 66 kbp, we have shown that OM as applied by Fluorocode is capable of detecting elements as short as 26 kbp .…”

“…26,27 In silico work by our group has produced interesting results regarding the identification of closely related strains, as well as the identification of relatively shorter genomic elements such as the SCCmec. 27 The bacteriophage genomes are between 40 and 70 kbp, while bacterial genomes are typically 1−4 Megabase pairs in length. It is of note that the scale of genomic elements to consider is different in OM compared to sequencing where the typical lengths are, for example, reads of 150 base pairs or amplicons of up to 1000 base pairs for PCR.…”

“…The experimentally obtained signal traces are compared to a database of in silico-generated traces. 26,27 The database is curated to contain a set of reference species of interest, in this case, the SCCmec elements I−IV. The "bars" of the barcode are preprocessed: each trace is locally normalized within a sliding window of 10 kb to avoid biassing by local changes in label density.…”

“…We have previously demonstrated by means of simulation that Fluorocode has the theoretical capacity to identify the exact SCCmec present in an S. aureus sample. 27 In this work, we showcase how Fluorocode performs in relation to real samples from cultures of S. aureus. Subsequently, we show the identification of MRSA DNA in complex mixtures of bacteria.…”

“…The Fluorocode methodology is described in detail in Bouwens et al and D’Huys et al , The method relies on the natural functionality of the methyltransferase enzyme M.Taq, originally isolated from the thermophilic bacterium Thermus aquaticus . In native environments, this enzyme methylates DNA, with S-adenosylmethionine (SAM) serving as the methyl donor.…”