Long-Read Plasmid Sequencing and Assembly Using Nanopore Sequencing-Based Workflows

We investigate the replication, maintenance, and dissemination of self-replicating circular DNA elements, known as plasmids, which necessitates the determination of their sequences. This protocol addresses coverage and accuracy issues associated with long-read sequencing by enriching plasmid DNA and optimizing assembly methods. Our protocol is designed to sequence plasmids in situations where the chromosomal sequence is either known or irrelevant.

To begin, streak the samples onto LB auger plates supplemented with the appropriate antibiotic to select for the respective plasmid-borne resistance marker. Incubate the plates at 37 degrees Celsius for 24 hours. The next day, inoculate three to five milliliters of LB medium supplemented with the appropriate antibiotic with a single isolated colony.

Incubate the culture with shaking at 37 degrees Celsius for approximately eight hours or until the optical density at 600 nanometers reaches one to two. Then inoculate 400 milliliters of LB medium supplemented with the appropriate antibiotic with 400 microliters of the starter culture and incubate as demonstrated earlier. Then transfer the culture into centrifuge bottles appropriate for the culture volume.

Centrifuge at 4, 200 G for 10 minutes and resuspend the pellet using the reagent volumes recommended by the manufacturer. Following cell lysis and neutralization, centrifuge the lysate at 4, 200 G for 30 minutes. Transfer the supernatant to a new tube and centrifuge it again.

Perform equilibration, washing, and DNA elution according to the manufacturer's instructions. Then resuspend the precipitated DNA in 100 microliters of nuclease-free water to maximize concentration for nanopore library preparation. Next, to quantify the DNA, prepare the assay tubes as per the presented table.

After calibrating the fluorometer, insert the sample tubes into the fluorometer and record the readings according to the instrument manual. Program the thermal cycler to 30 degrees Celsius for two minutes, followed by 80 degrees Celsius for two minutes. Spin and mix the thawed kit components as per the presented table.

Incubate the PCR tubes prepared for barcoding using the thermal cycler program. Then place the tubes on ice to cool. Pool the barcoded samples in a clean 1.5 milliliter Eppendorf tube, ensuring the total volume does not exceed 1, 000 microliters.

Once the beads are vortexed, add an equal volume of beads to the pooled barcoded samples. Mix by flicking the tube, followed by a 10-minute incubation. Then wash the beads with one milliliter of 80%ethanol while on the magnet.

Remove the ethanol and repeat once. Remove the tube from the magnet. Resuspend the beads in elution buffer using the appropriate volume based on the number of barcodes in use and incubate for 10 minutes at room temperature.

Quantify the DNA concentration using one microliter of the eluded sample on the fluorometer to confirm successful library preparation. For adapter ligation, transfer 11 microliters of the DNA library into a new tube and label it. Then mix the presented components in a fresh tube to dilute the thawed rapid adapter.

Next, add one microliter of diluted rapid adapter to the 11 microliters of DNA library and flick the tube to mix the contents. Spin down the tube briefly to ensure the complete volume collects at the bottom and incubate the tube on the bench for five minutes. Prepare the flow cell priming mix in a fresh DNA tube and mix by inversion and pipetting.

Lift the lid of the device and insert the flow cell into it while applying gentle pressure to ensure proper placement. After checking for air bubbles within or adjacent to the priming port, draw back 20 to 30 microliters of fluid to remove bubbles. Then draw up 800 microliters of priming mix and dispense it into the priming port while avoiding air bubbles and let the device sit for five minutes.

Mix the DNA library with the appropriate volume of library beads in a new tube as presented here. Load 200 microliters of priming mix into the flow cell priming port. Adjust the sample port cover to reveal the port.

Dispense 75 microliters of the DNA library with beads into the sample port one drop at a time, allowing each drop to absorb before adding the next. Then reapply the sample port cover and close the priming port. Once the device lid is closed, begin the sequencing run.

Set the runtime limit based on the number of plasmids being multiplexed with a default of 48 hours and a maximum of 72 hours. Select the super accurate base calling mode and the appropriate rapid barcode kit option. Then select the raw FAST5 or POD5 data and start base calling.

To generate a filtered FASTQ file for downstream assembly, trim long reads using Filtlong to remove approximately 5-10%of the lowest quality reads while retaining approximately 90%of the total yield. Subsample reads using Autocycler according to recommended settings. Assemble each read subset using Raven, Flye, Kanu, Miniasm, Myloasm, NECAT, Plassembler, and other selected assemblers.

Visualize each assembly graph file using bandage. Evaluate contigs for circularity, size consistency across assemblers, and evidence of chimeric joints. Remove linear contigs lacking support across multiple assemblers and retain circular contigs with consistent size estimates.

Remove minor unsupported contigs before consensus generation when major plasmids are circularized. Proceed to consensus generation after manual curation. Generate the consensus sequence using Autocycler with the curated assemblies.

Execute the remaining Autocycler commands to produce the final consensus plasmid sequence. Lastly, polish the final assembly using trimmed long reads with Medaka to improve consensus accuracy. All conjugation assays produced transconjugants, confirming the presence of a conjugative plasmid in the donors.

The sizes of assembled plasmid sequences were congruent with estimates based on pulse field gel electrophoresis mobility. Nanopore sequencing produced sufficient read output and quality for all samples analyzed. Chromosomal contamination rates ranged between 72.1%and 28.0%with an average of 46.9%Pulse field gel electrophoresis analysis of strain plasmid UPEC_271 revealed a band corresponding to a plasmid of approximately 145 kilobase pairs.

DNA from one of the transconjugants showed a sequence identical to the 145 kilobases. Pulse field gel electrophoresis analysis of strain blood 100913 identified two plasmids of approximately 70 and 110 kilobase pairs. Nanopore sequencing confirmed two plasmid assemblies of 71, 182 base pairs and 111, 226 base pairs.

The most important consideration is whether chromosomal sequence is of interest. If so, standard whole genome sequencing protocols are better. Our protocol's increased coverage opens the door to the study of heteroplasmy, i.e.

plasmid sequence variation within a given population.