Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Abstract

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

“…The work expands on previous in vitro studies of large DNA molecules. 29 , 32 , 34 , 50 , 51 , 52 For example, Wegner et al. 30 , 49 and Cunha et al.…”

“…Several previous studies have proposed methods to extract chromosomes from cells, and some have even used protein-removal steps to obtain deproteinated DNA. 29 , 30 , 31 , 32 , 33 , 34 However, most of these studies lacked an imaging-based characterization of the resulting DNA objects regarding their size, level of deproteination, and suitability for in vitro imaging-based experiments.…”

DNA in nanochannels: theory and applications

“…As a result, the emission intensity will be bright in GC-rich regions, and dark in AT-rich regions, along the extended DNA molecule. One advantage of denaturation mapping is that it can be done either on-chip (Reisner et al ., 2010; Welch et al ., 2012) or prior to sample loading (Marie et al ., 2013; Łopacińska-Jørgensen et al ., 2017). The most recent studies using denaturation mapping have been focused on stretching very long DNA molecules by hydrodynamic flow in a shallow nanoslit.…”

Consistent ultra-long DNA sequencing with automated slow pipetting

“…The “nuclei-method” later expanded on this technique by enriching cell nuclei before embedding in agarose to remove cytoplasmic and other cellular lysates from the resulting DNA [18]. Second-generation short read sequencing can be performed on DNA extracted using the nuclei method [19]. However, gel extraction protocols designed for preserving high molecular weight DNA sequences require access to specialized electroelution chambers or pulsed-field gel electrophoresis systems [20].…”