Oxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA)

Measure the concentration of each dMDA sample using the Qubit Fluorometer. Mix 1µL of DNA with 199µL of 1X dsDNA HS reagent, in a 0.5 ml Qubit tube. Vortex for 0h 0m 10s, spin briefly in a microfuge and incubate at Room temperature for 0h 2m 0s and measure concentration.

(Optional) Assess the DNA integrity and fragment-length profile of each sample using the Agilent TapeStation and Genomic ScreenTape Assay. Use 1µL of DNA, at a concentration of 10-100ng/µL for TapeStation analysis.

Aliquot 500ngof each dMDA sample into a 0.2 ml thin-walled PCR tube and make the volume up to 26µL with nuclease-free water.

Prepare the following reaction:

Incubate reactions in a thermal cycler at 37°C for 1h 0m 0s

Prepare custom SPRI beads , by replacing the buffer of AMPure XP beads with custom AMPure XP buffer (according to the ONT protocol: Ligation sequencing gDNA – whole genome amplification (SQK-LSK110) Version: WAL_9115_v110_revH_10Nov2020).

Mix AMPure XP beads by vortexing thoroughly and transfer two 1 ml aliquots into two 1.5 ml Eppendorf LoBind tubes.

Place tubes in a magnetic rack for 0h 2m 0s, to pellet the beads and then remove the supernatant.

Remove the tubes from the magnet and resuspend the beads by adding 1mL of nuclease-free water and vortexing, then return the tubes to the magnet rack.

Repeat the above wash step, for a total of 2 washes.

Briefly centrifuge the tubes, return to the magnet, and use a P20 pipette to remove any remaining water.

Resuspend and pool the two pellets in 200µL of custom AMPure buffer, then transfer the resuspended beads to the 2 ml tube containing the remaining custom AMPure buffer.

Vortex thoroughly and make sure beads are thoroughly mixed again just before use.

Adjust debranching reactions to 50µL using TE buffer (pH 8), add 35µL of custom SPRI beads and incubate on a Hula-Mixer at 9rpm.

Pellet beads on a magnetic rack for 0h 2m 0s, or until beads are completely separated from solution and remove the supernatant.

Leave tubes on the magnet and wash the pellet by carefully adding 200µL of 70 % ethanol. Do not disturb the bead pellet and remove ethanol after 0h 0m 30s.

Repeat the above wash step, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

Air-dry pellet for up to 0h 2m 0s.

To elute DNA from the beads, resuspend the pellet by adding 20µL of nuclease-free water and flicking the tubes to thoroughly resuspend the beads.

Briefly spin the tubes in a microfuge for 1-2 s.

Incubate in a thermal cycler at 50°C for 0h 1m 0s.

Incubate at Room temperature for 0h 9m 0s.

Pellet beads on a magnetic rack for 0h 2m 0s.

Transfer the supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

Quantify 1µL of eluted DNA by Qubit.

The following library preparation steps follow the protocol Ligation sequencing gDNA – Native Barcoding Kit 24 V14; Version: NBE_9169_v114_revQ_15Sep2022, with some modifications. For each sample, use all recovered debranched dMDA DNA from step 18.

End-prep:

Prepare the following reaction for each sample, in a 0.2 ml thin-walled PCR tube:

Mix samples by pipetting and briefly centrifuge.

Incubate reactions in a thermal cycler at 20°C for 0h 30m 0s, then 65°C for 0h 5m 0s.

Mix AMPure XP beads by vortexing and add 60µL to each reaction.

Mix the AMPure beads with the reaction solution by thoroughly flicking and inverting the tubes.

Incubate at Room temperature for 0h 5m 0s.

Briefly centrifuge the tubes then place on a magnetic rack for ~ 0h 2m 0s, or until the beads have completely pelleted.

Remove the supernatant.

Leave tubes on the magnet and wash the pellet by carefully adding 200µLof 80 % ethanol. Do not disturb the bead pellet and remove ethanol after 0h 0m 30s.

Repeat the above wash step, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

Air-dry pellet for up to 0h 2m 0s.

To elute DNA from the beads, remove tubes from the magnetic rack and resuspend the pellets by adding 23µL of nuclease-free water and flicking the tubes to thoroughly resuspend the beads. Briefly spin the tubes in a microfuge for 1-2 s and incubate in a thermal cycler at 37°C for 0h 10m 0s.

Pellet beads on a magnetic rack for 0h 2m 0s and then transfer 22.5µL of supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

Barcode Adapter Ligation:

Prepare the following barcode adapter ligations at Room temperature

Gently pipette mix to thoroughly mix reactions and briefly centrifuge.

Incubate at Room temperature for 0h 30m 0s.

Mix AMPure XP beads by vortexing and add 100µL to each reaction.

Mix the AMPure beads with the reaction solution by thoroughly flicking and inverting the tubes and incubate at Room temperature for 0h 5m 0s.

Briefly centrifuge the tubes then place on a magnetic rack for ~ 0h 2m 0s, or until the beads have completely pelleted.

Remove the supernatant.

Leave tubes on the magnet and wash the pellet by carefully adding 200µL of 80 % ethanol. Do not disturb the bead pellet and remove ethanol after 0h 0m 30s.

Repeat the above wash step, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

Air-dry pellet for up to 0h 2m 0s.

To elute DNA from the beads, remove tubes from the magnetic rack and resuspend the pellets by adding 16µL of nuclease-free water and flicking the tubes to thoroughly resuspend the beads. Briefly spin the tubes in a microfuge for 1-2 s and incubate in a thermal cycler at 37°C for 0h 10m 0s.

Pellet beads on a magnetic rack for 0h 2m 0s and then transfer the supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

Quantify 1µL of eluted DNA by Qubit.

Sequencing Adapter Ligation:

Combine barcoded samples in equal amounts, in a 1.5 ml LoBind tube, and use the pooled sample to prepare the below reaction. Add reagents in the listed order:

Incubate ligation reaction at Room temperature for 0h 30m 0s.

Add 100µL of resuspended AMPure beads and incubate on a HulaMixer for 0h 10m 0s at 9rpm,0h 0m 0s.

Briefly centrifuge the tube then place on a magnetic rack for ~ 0h 2m 0s, or until the beads have completely pelleted.

Remove the supernatant.

Remove tube from the magnetic stand and wash the beads by adding 125µL of Short Fragment Buffer (SF) and thoroughly resuspend beads by flicking the tube.

Return tube to the magnetic stand and pellet beads for ~ 0h 2m 0s.

Repeat the above wash steps, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining SFB.

Air-dry pellet for up to 0h 0m 30s.

To elute DNA from the beads, remove the tube from the magnetic rack and resuspend the pellet by adding 25µL of Elution Buffer (EB) and flicking the tube to thoroughly resuspend the beads.

Briefly spin the tube in a microfuge for 1-2 s and incubate in a heat block or water bath at 37°C for 0h 10m 0s.

Pellet beads on a magnetic rack for 0h 2m 0s and then transfer the supernatant, containing eluted DNA, to a new 1.5 ml LoBind tube.

Quantify 1µL of eluted DNA by Qubit.

Prepare and load PromethION flow cell according to the protocol: Ligation sequencing gDNA – Native Barcoding Kit 24 V14; Version: NBE_9169_v114_revQ_15Sep2022 .

Start run in MinKNOW using default parameters for the NBD114.24 or NBD114.96 library prep kit.

Monitor number of available pores during the run and perform nuclease flush and reloads as needed, using the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) and the protocol: Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) Version: WFC_9120_v1_revQ_08Dec2020 . A 72h 0m 0s run will normally require 1-2 flush and reloads to maximise the output of the flow cell.