μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome

Abstract

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.

A tunable filter for high molecular weight DNA selection and linked-read sequencing

“…After size selection operations with the mono-or multicapillary systems, the different sample fractions were characterized with the µLAS separation and titration protocol described in 28 . Sizing and quantification errors are 3% and 10%, respectively.…”

“…Because DNA molecules are not entrapped in the separation matrix, they can subsequently be sorted and purified by size, as recently reported with a selection cut-off of ~2 kbp 18 . Alternatively, we recently developed the "µ-Laboratory for DNA Analysis and Separation" (µLAS) technology for DNA analysis in the size range 0.1 to 200 kbp 19,20 . This technology is operated in a capillary electrophoresis system by controlling the fluid flow and using a counter electrophoretic force 21 .…”

“…This technology is operated in a capillary electrophoresis system by controlling the fluid flow and using a counter electrophoretic force 21 . The key component of µLAS is a constriction, in which DNA can be concentrated before its analysis with a limit of detection of 10 fg/µL 19,20,22 . Because the technology is operated without separation matrices, it can be used for DNA size selection 20 .…”

“…The key component of µLAS is a constriction, in which DNA can be concentrated before its analysis with a limit of detection of 10 fg/µL 19,20,22 . Because the technology is operated without separation matrices, it can be used for DNA size selection 20 . Here, we set out the development of a tunable size selection filter to rapidly produce 10 to 20 ng of DNA for TGS.…”

Single-step electrohydrodynamic separation of 1–150 kbp in less than 5 min using homogeneous glass/adhesive/glass microchips

“…Alternatively, free solution DNA separation based on hydrodynamic actuation in narrow capillaries of ~2 µm enabled the fractionation of molecules of up to 100 kbp in one hour (Wang et al 2012). The combination of electrophoresis and hydrodynamics acting in opposite directions, hereafter called electrohydrodynamic migration, also appeared as a free solution separation technology efficient for long molecules, as initially reported with 5 and 50 kbp DNA (Zheng and Yeung 2002) and recently improved in the range of 3 to 200 kbp with a separation time of one hour (Milon et al 2019). In order to consolidate the promising performances of electrohydrodynamic migration, which relies on the existence of size-dependent transverse forces that set molecules close to the walls (Ranchon et al 2016) (ADD here 2 papers from Butler), we wished to test whether microchips would allow us to gain time and extend the separation range.…”