Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap

Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA’s electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA’s negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.


Control of HMW DNA Quality and Integrity
The ratio between absorbance at 260 nm and at 230 nm (A260/A230) was calculated to estimate DNA purity.The absorbance values were determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific).HMW DNA concentrations measured using NanoDrop were validated with a Qubit 3.0 fluorometer (Invitrogen) using the dsDNA BR assay kit (Thermo Fisher Scientific).DNA integrity was assessed by electrophoresis on 1% agarose gels.

Long-Read Sequencing with Oxford Nanopore Technologies (ONT)
Sequencing libraries were prepared using the Rapid Barcoding kit (SQK-RBK110.96,Oxford Nanopore Technologies) and the solid-phase reversible immobilization (SPRI) beads for DNA clean-up following the manufacturer's instructions.Duplicate runs with different barcodes were performed for each sample, and a single sample was pooled for sequencing on a single flow cell.Each library containing ~0.5 μg of DNA was sequenced using the ONT GridION platform with R9.4.1 chemistry.Sequencing ran for 25 hours until the flow cell buffer was exhausted.Data acquisition, real-time analysis, and sample tracking were carried out using the MinKNOW (v.2.1) software.High-accuracy base calling was performed from the fast5 files using the Oxford Nanopore Guppy tool (v.3.0.4).The run was monitored using RAMPART (https://github.com/articnetwork/rampart),enabling it to stop the run once a minimum sequencing depth of 20x was achieved.

Sodium Content Measurements
Sodium content measurements were carried out by inductively coupled plasma optical emission spectroscopy (ICP-OES) using an Agilent 720 instrument (Agilent Technologies, Inc., Santa Clara, CA, USA).The following sodium atomic lines were selected: 568.821 nm, 588.995 nm and 589.592 nm.The average sodium content was calculated from two replicates.Prior to analysis, all samples were diluted 20fold with ultrapure water.The accuracy was estimated using spiked sample standards following the ISO 5725-4 (2020) guidelines.

Supplementary Protocols Preparation of 50X THE Running Buffer (1 M Tris, 1 M HEPES, 5 mM EDTA (optional), pH 8.0).
Dissolve 121,14 g Tris-base, 238,3 g HEPES (free acid) in MilliQ water, add 10 ml 0.5 M EDTA (optional), and bring the final volume to 1 litter.The pH does not need to be adjusted and should be between 8.0 and 8.1.

DNA Extraction Using the SDS/Proteinase K Method
This protocol is used to extract crude DNA for gel loading from complex plant, animal, insect, fungal, and microbial samples.The following example shows how to extract DNA from a 100 µg leaf sample.

1.
Grind the deep-frozen leaf tissue in liquid nitrogen with a pre-chilled mortar and pestle.Transfer the ground powder to a 1.5 ml microtube.

3.
Transfer a 50 µl aliquot of the lysate to a new 1.5 ml microtube and mix with loading buffer as described in step 7 of the gel purification protocol below.Freeze the remaining lysate until further use.

DNA Extraction Using the CTAB Method
This protocol is used to extract crude DNA for gel loading from complex samples (e.g., soil, feces, wood) containing a high concentration of organic compounds such as humic substances, pigments, etc.The example below shows how to extract DNA from samples weighing 1 to 5 grams, but the protocol can be scaled down accordingly.

2.
Centrifuge the homogenate at 14,000 x g for 10 minutes at +4С.While the centrifugation is in progress, prepare two new 50-ml Falcon tubes containing 20 ml of 100% isopropanol chilled to -20С.

3.
Divide the clarified supernatant into two equal 20-ml portions and transfer them to the Falcon tubes containing isopropanol.Vortex thoroughly.

5.
Discard the supernatant without disturbing the DNA pellet.

6.
Wash the pellet in each tube by adding 10 ml of 70% ethanol.Vortex briefly.

8.
Completely remove the supernatant, but do not dry the pellet.Add 1 ml of 1 x TE (10 mM Tris-HCl, 1 mM Na 2 EDTA, pH 8.0) to the pellet, vortex, and incubate at 65С with occasional mixing until the pellet is completely dissolved.Pool the DNA solution from both Falcon tubes into one 2-ml microtube.

9.
Transfer a 50 µl aliquot of the DNA solution to a new 1.5 ml microtube and mix with loading buffer as described in step 7 of the gel purification protocol below.Freeze the remaining DNA until further use.

ONT Sequencing Protocol for GridION Instrument
DNA concentration before starting was 17,0 ng/l (as measured by Qubit).

2.
Add 5 µl of Rapid Barcode Plate for each.

6.
Add an equal volume of resuspended SPRI.