Extraction and ONT MinION Library Preparation of uHMW gDNA
Kaylee S. Herzog, jfauver
Abstract
This custom protocol optimizes extraction, purification, and Oxford Nanopore Technologies (ONT) MinION library preparation for ultra-high molecular weight genomic DNA (uHMW gDNA) from parasitic nematodes. It can be used effectively with both low-input samples (e.g., a single adult hookworm) and high-input samples (e.g., a chunk of tissue from an Ascaris sp. adult).
Protocols on which this workflow is based:
Before start
For new kits, add 1,040 µL Zymo Proteinase K Storage Buffer to each tube of Zymo Proteinase K (20 mg) prior to use. The final concentration of Proteinase K is ~20 mg/ml. Store resuspended Proteinase K at -20°C after mixing.
Steps
Part 1: Ultra-HWM gDNA extraction | Zymo Quick-DNA HWM MagBead Plus Kit | ~3 hr
Set dry bath to 55°C
For each sample, add the following to a clean 1.5 mL microcentrifuge tube to create a master mix:
`95µL` <reagents text="Zymo DNA Elution Buffer" label="Zymo Research"/>
`95µL` <reagents text="Zymo Biofluid & Solid Tissue Buffer" label="Zymo Research"/>
`10µL` <reagents text="Zymo Proteinase K" label="Zymo Research"/>
Vortex the master mix gently to mix, then spin down and keep on ice
Using a new pipette tip or sterilized forceps, add one whole worm (or a piece of tissue) directly from tissue preservative to the bottom of a clean 1.5 mL microcentrifuge tube
Use a new
Add 200µL master mix (prepared in Part 1 Step 2) to each tube containing tissue and pestle
Continue using the pestle to grind the tissue within the master mix until fully homogenized. Remove the pestle, being careful to keep any tissue in the tube by wiping the pestle on the tube edges as it is removed
Close the tube and mix by inverting and flicking gently, then spin down briefly to recollect tissue and liquids
Incubate sample in dry bath at 55°C for 2h 30m 0s or until tissue solubilizes. During incubation, flick tube every 0h 20m 0s to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
Set dry bath to 37°C
Add 400µL
Flick tubes to mix, then spin down briefly to recollect liquids
Add 33µL
To ensure DNA binds to beads, mix on a rotator mixer at a low speed for 1h 30m 0s at Room temperature. Spin down briefly before proceeding with the next step
Set sample tubes on a magnetic stand until beads have separated from solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand.
Add 500µL
Flick to mix initially, then mix on a rotator mixer at a low speed for 0h 20m 0s at Room temperature. Spin down briefly before proceeding with the next step
Set sample tubes on a magnetic stand until beads have separated from the solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand
Add 500µL
Flick to mix, then spin down briefly
Set sample tubes on a magnetic stand until beads have separated from solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand
Add 900µL
Flick to mix, then spin down briefly
Transfer the entire sample (all liquid and beads) to a new clean 1.5 mL microcentrifuge tube
Set samples (now in new tubes) on a magnetic stand until beads have separated from the solution, then remove and discard the supernatant. Remove sample tubes from the magnetic stand
Add 900µL
Flick to mix, then spin down briefly
Transfer the entire sample (all liquid and beads) to a new clean 1.5 mL microcentrifuge tube
Set samples (now in new tubes) on a magnetic stand until beads have separated from the solution, then remove and discard the supernatant. Leave sample tubes on the magnetic stand
Use a P10 pipette to remove any residual liquid from the bottom of the tube
Air dry the beads for up to 0h 20m 0s and proceed to next step once beads are dry, but not over-dry
Add 50µL
Incubate in dry bath at 37°C for 2h 0m 0s. During incubation, flick tube every 0h 20m 0s to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
Incubate on bench top at Room temperature overnight.
After overnight incubation, set tubes on a magnetic stand until beads have separated from solution, then move the supernatant (now containing eluted gDNA) to a new clean 1.5 mL microgentrifuge tube
Re-suspend beads in 20µLof
Use 1µL of final elution to quantify extraction via Qubit analysis
Use 1µL of final elution to assess fragment size distribution via TapeStation
Part 3: DNA repair and end-prep | Zymo Clean & Concentrator, ONT Ligation Sequencing, & NEBNext Companion Kits | ~1.5 hr
Set dry bath to 65°C
Defrost the needed NEB DNA and End Repair reagents on ice (see Part 3 Step 38)
For each sample, add the following to a clean 0.2 mL PCR tube to create a master mix, pipetting 10–20 times between each addition to mix:
`3.5µL` <reagents text="NEBNext® FFPE DNA Repair Buffer" label="New England Biolabs"/>
`2µL` <reagents text="NEBNext FFPE DNA Repair Mix - 96 rxns" label="New England Biolabs"/>
`3.5µL` <reagents text="NEBNext Ultra II End Prep Reaction Buffer" label="New England Biolabs"/>
`3µL` <reagents text="NEBNext Ultra II End Prep Enzyme Mix" label="New England Biolabs"/>
Keep master mix on ice
Add 12µL of master mix (prepared in Part 3 Step 38) from the PCR tube directly into each 1.5 mL microcentrifuge tube containing extracted & purified uHWM gDNA (from Part 2). Mix all components by gently flicking, and spin tubes down to recollect liquids
Incubate samples at Room temperature for 0h 10m 0s
Incubate samples in dry bath at 65°C for 0h 10m 0s
Add 4 volumes of
Spin samples down briefly and add 20µL
Mix samples on rotating mixer at a low speed at Room temperature for 1h 30m 0s
Briefly spin down samples and pellet on a magnetic stand (1–2 min) until the supernatant is clear and colorless. With the tubes still on the magnet, pipette off and discard the supernatant
Add 500µL
Briefly spin samples down briefly and transfer to magnetic stand to allow beads to pellet until solution is clear (1–2 min). With the tubes still on the magnet, pipette off and discard the supernatant
Add 500µL
Briefly spin samples down briefly and transfer to magnetic stand to allow beads to pellet until solution is clear (1–2 min). With the tubes still on the magnet, pipette off and discard the supernatant
Air dry the beads for 0h 10m 0s
Add 51µL
Manually agitate samples for 0h 10m 0s by gently flicking/inverting (and occasionally spinning dow to recollect liquids)
Briefly spin samples down and pellet the beads on a magnet until the eluate is clear and colorless (1–2 min)
Remove and retain the 51µL of eluate (containing repaired & end-prepped DNA) to a new clean 1.5 mL microcentrifuge tube
Use 1µL of final elution to quantify via Qubit assay
Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing & NEBNext Companion Kits | ~3 hr + overnight incubation
Set dry bath to 37°C
Remove 4°C and allow them to come to Room temperature
Spin down
Thaw Room temperature, spin down, and mix by pipetting. Place on ice immediately after thawing and mixing
Thaw Room temperature, vortex to mix, spin down, and place on ice
Thaw one tube each of Room temperature, vortex to mix, spin down, and place on ice
For each sample, add the following, in order, to a new clean 1.5 mL microcentrifuge tube, pipetting 10–20 times between each addition to mix:
`25µL` <reagents text="ONT Ligation Buffer (LNB)" label="Oxford Nanopore Technologies"/>
`10µL` <reagents text="Quick T4 DNA Ligase" label="New England Biolabs"/>
`5µL` <reagents text="ONT Adaptor Mix (AMX)" label="Oxford Nanopore Technologies"/>
Keep master mix on ice after mixing
For each sample, prepare 1:3 SFB:LFB titrated wash mix by adding the following to a new clean 1.5 mL microcentrifuge tube, and then vortex to mix:
`125µL` <reagents text="Short Fragment Buffer (SFB)" label="Oxford Nanopore Technologies"/>
`375µL` <reagents text="Long Fragment Buffer (LFB)" label="Oxford Nanopore Technologies"/>
Keep titrated wash mix on ice after vortexing
Pipette 40µL of master mix (prepared in Part 4 Step 62) directly into entire volume of repaired and end-prepped gDNA from Part 3. Mix all components by gently flicking and spin tube down to recollect liquids
Incubate the reaction 0h 15m 0s at Room temperature
Resuspend
Mix on a rotator mixer at a low speed for 1h 0m 0s at Room temperature
Spin down the sample and pellet on a magnetic stand. Keeping the tube on the stand, pipette off and discard the supernatant
Wash the beads by adding 250µL 1:3 SFB:LFB titrated wash mix (prepared in Part 4 Step 63). Flick the beads to resuspend, spin down, then return to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard
Wash the beads by adding 250µL 1:3 SFB:LFB titrated wash mix (prepared in Part 4 Step 63). Flick the beads to resuspend, spin down, then return to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard
Spin down the beads and place them back on the magnetic rack. Use a P10 pipette to pipette of any residual liquid and allow beads to air-dry for 0h 0m 30s to 0h 2m 0s
Remove the tube from the magnetic stand and resuspend the beads in 15µL
Briefly spin down and incubate in dry bath at 37°C for 2h 0m 0s. During incubation, flick tube every 0h 20m 0s to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
Incubate on the bench top at Room temperature overnight
After overnight incubation, pellet the beads on a magnet until the eluate is clear and colorless (at least 1 min)
Remove and retain the 15µL of eluate (containing the prepared library) to a new clean 1.5 mL microcentrifuge tube
Use 1µL of final elution to quantify library via Qubit analysis
Use 1µL of final elution to assess fragment size distribution via TapeStation
