Jul 19, 2023

Public workspaceExtraction and ONT MinION Library Preparation of uHMW gDNA V.5

DOI
dx.doi.org/10.17504/protocols.io.j8nlkww11l5r/v5
  • 1University of Nebraska Medical Center
Kaylee S. Herzog
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkww11l5r/v5
Protocol Citation: Kaylee S. Herzog, jfauver 2023. Extraction and ONT MinION Library Preparation of uHMW gDNA. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkww11l5r/v5Version created by Kaylee S. Herzog
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 18, 2023
Last Modified: July 19, 2023
Protocol Integer ID: 85191
Keywords: extraction, gDNA, MinION, nematode, library preparation, uHMW gDNA, ONT
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:
Materials
  • Oxford Nanopore Technologies Ligation Sequencing Kit V14 (SQK-LSK-114)
  • Zymo Quick-DNA Magbead Plus Kit (D4081)
  • Zymo DNA Clean & Concentrator Magbead Kit (D4012)
  • NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (E7180S)
  • NEB Monarch Pestle (one per specimen; T3000L)
  • AMPure XP strepatavidin beads (A63880)
  • Minicentrifuge
  • Micropipetters & tips (wide-bore tips STRONGLY recommended for pipetting up gDNA or libraries)
  • Dry bath
  • Rotator mixer (e.g., PR1MA Roto-Mini Mixer)
  • Magnetic separation rack (e.g., MagJET Separation Rack)
  • 1.5 mL microcentrifuge tubes (DNase-free, DNA low bind STRONGLY recommended)
  • 0.2 mL PCR tubes
  • Microcentrifuge (only if processing large [i.e., >2 cm] specimens)
Protocol materials
ReagentZymo Quick-DNA™ MagBinding Buffer Zymo ResearchCatalog #D4077-1-150
In 2 steps
ReagentAMPure XP BeadsBeckman CoulterCatalog #A63880
In 2 steps
ReagentZymo Proteinase KZymo ResearchCatalog #D3001-2-20
Step 2
ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies
Step 64
ReagentNEBNext FFPE DNA Repair Mix - 96 rxnsNew England BiolabsCatalog #M6630L
Step 38
ReagentZymo DNA MagBinding BufferZymo ResearchCatalog #D4012-1-50
Step 42
ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
Step 38
ReagentZymo g-DNA Wash BufferZymo ResearchCatalog #D3004-2-200
In 2 steps
ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-50
Step 30
ReagentNuclease-free Water
Step 33.1
ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
Step 38
ReagentONT Ligation Buffer (LNB)Oxford Nanopore Technologies
Step 60
ReagentZymo DNA Pre-Wash BufferZymo ResearchCatalog #D3004-5-250
Step 18
ReagentNEBNext® FFPE DNA Repair BufferNew England BiolabsCatalog #E7180S
Step 38
ReagentZymo MagBinding BeadsZymo ResearchCatalog #D4100-2-6
Step 12
ReagentZymo DNA Wash BufferZymo ResearchCatalog #D4003-2-24
In 2 steps
ReagentLigation Adaptor (LA)Oxford Nanopore Technologies
In 3 steps
ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Step 2
ReagentZymo Biofluid & Solid Tissue BufferZymo ResearchCatalog #D4081-3-25
Step 2
ReagentElution Buffer (EB)Oxford Nanopore Technologies
Step 61
ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies
Step 62
ReagentLong Fragment Buffer (LFB)Oxford Nanopore Technologies
In 2 steps
ReagentElution Buffer (EB)Oxford Nanopore Technologies
Step 73
ReagentZymo MagBinding BeadsZymo ResearchCatalog #D4100-5-2
Step 43
ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-10
Step 51
ReagentQuick T4 DNA LigaseNew England BiolabsCatalog #E7180S
In 2 steps
ReagentNEB Monarch PestleNEBCatalog #T3002-1
Step 4
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.
Part 1: Ultra-HWM gDNA extraction | Zymo Quick-DNA HWM MagBead Plus Kit | ~3 hr
Part 1: Ultra-HWM gDNA extraction | Zymo Quick-DNA HWM MagBead Plus Kit | ~3 hr
Set dry bath to Temperature55 °C

For each sample, add the following to a clean 1.5 mL microcentrifuge tube to create a master mix:
Amount95 µL ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-1
Amount95 µL ReagentZymo Biofluid & Solid Tissue BufferZymo ResearchCatalog #D4081-3-25
Amount10 µL ReagentZymo Proteinase KZymo ResearchCatalog #D3001-2-20

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
Note
Transfer as little tissue preservative liquid as possible to the new tube during this process

Use a new ReagentNEB Monarch PestleNEBCatalog #T3002-1 to grind and crush the tissue in the tube. Keep the pestle in the tube

Add Amount200 µL 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 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 Temperature55 °C for Duration02:30:00 to DurationOvernight until tissue solubilizes. During incubation, flick tube every Duration00:20:00 to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
Note
If a very large amount of input tissue was used: It is likely there will still be visible tissue even after hours of lysis. If so, centrifuge the sample for Duration00:01:00 at Centrifigation10000 x g or greater to pellet debris, then pipette all liquids into a new clean 1.5 µL microcentrifuge tube. (The majority of gDNA will be contained in the layer of liquid just above the pellet, so pipette carefully to get as much liquid as possible without disturbing the debris.) Discard the tube contain the pelleted debris and use the retained supernatant for Part 2.




3h 10m
Incubation
Digestion
Overnight
Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
Part 2: Ultra-HWM gDNA purification | Zymo Quick-DNA HWM MagBead Plus Kit | ~4 hr + overnight incubation
2h 20m
2h 20m
Set dry bath to Temperature37 °C

Add Amount400 µL ReagentZymo Quick-DNA™ MagBinding Buffer Zymo ResearchCatalog #D4077-1-150 to each sample

Flick tubes to mix, then spin down briefly to recollect liquids
Add Amount33 µL ReagentZymo MagBinding BeadsZymo ResearchCatalog #D4100-2-6 to each sample
Note
MagBinding Beads settle quickly, so ensure beads are kept in suspension while dispensing by vortexing the beads each time before they are added to a sample


To ensure DNA binds to beads, mix on a rotator mixer at a low speed for Duration02:00:00 at TemperatureRoom temperature . Spin down briefly before proceeding with the next step

2h
Mix
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.
Note
Some beads may adhere to the sides of the tube. When removing supernatant, aspirate slowly to allow these beads to be pulled to the magnet as the liquid level is lowered.

Add Amount500 µL ReagentZymo Quick-DNA™ MagBinding Buffer Zymo ResearchCatalog #D4077-1-150 to each sample
Flick to mix initially, then mix on a rotator mixer at a low speed for Duration00:20:00 at TemperatureRoom temperature . Spin down briefly before proceeding with the next step

20m
Mix
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
Note
Some beads may adhere to the sides of the tube. When removing supernatant, aspirate slowly to allow these beads to be pulled to the magnet as the liquid level is lowered.

Add Amount500 µL ReagentZymo DNA Pre-Wash BufferZymo ResearchCatalog #D3004-5-250 to each sample
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
Note
Some beads may adhere to the sides of the tube. When removing supernatant, aspirate slowly to allow these beads to be pulled to the magnet as the liquid level is lowered.

Add Amount900 µL ReagentZymo g-DNA Wash BufferZymo ResearchCatalog #D3004-2-200 to each sample

Flick to mix, then spin down briefly
Transfer the entire sample (all liquid and beads) to a new clean 1.5 mL microcentrifuge tube
Note
Transfer to a new tube ensures that any salts that are stuck to the lid of the tube do not get carried over

Critical
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
Note
Some beads may adhere to the sides of the tube. When removing supernatant, aspirate slowly to allow these beads to be pulled to the magnet as the liquid level is lowered

Add Amount900 µL ReagentZymo g-DNA Wash BufferZymo ResearchCatalog #D3004-2-200 to each sample
Flick to mix, then spin down briefly
Transfer the entire sample (all liquid and beads) to a new clean 1.5 mL microcentrifuge tube
Note
Transfer to a new tube ensures that any salts that are stuck to the lid of the tube do not get carried over

Critical
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
Note
Some beads may adhere to the sides of the tube. When removing supernatant, aspirate slowly to allow these beads to be pulled to the magnet as the liquid level is lowered

Use a P10 pipette to remove any residual liquid from the bottom of the tube
Air dry the beads for up to Duration00:20:00 and proceed to next step once beads are dry, but not over-dry
Note
It may take less time for the beads to dry, so check them often during this process. Beads will change in appearance from glossy black when still wet to a matte black/brown when fully dry. Over drying the beads may result in lower gDNA recovery.

20m
Add Amount50 µL ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-50 to each sample and flick gently several times to mix. Spin down briefly


Note
If you plan to Qubit and TapeStation the extraction, it is a good idea to elute in 52 µL (rather than 50 µL) to have 1 µL easily available for each quality control analysis


Incubate in dry bath at Temperature37 °C for Duration02:00:00 . During incubation, flick tube every Duration00:20:00 to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
2h 20m
Incubation
Incubate on bench top at TemperatureRoom temperature overnight.

Incubation
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 microcentrifuge tube
Note
The eluted DNA can be used immediately or stored at Temperature4 °C or Temperature-20 °C for future use


Re-suspend beads in Amount20 µL of ReagentNuclease-free Water in case there is no (or not enough) gDNA in final elution

Use Amount1 µL of final elution to quantify extraction via Qubit analysis

Use Amount1 µL of final elution to assess fragment size distribution via TapeStation

Optional
Part 3: DNA repair and end-prep | Zymo Clean & Concentrator, ONT Ligation Sequencing, & NEBNext Companion Kits | ~1.5 hr
Part 3: DNA repair and end-prep | Zymo Clean & Concentrator, ONT Ligation Sequencing, & NEBNext Companion Kits | ~1.5 hr
1h
1h
Set dry bath to Temperature65 °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:
Amount3.5 µL ReagentNEBNext® FFPE DNA Repair BufferNew England BiolabsCatalog #E7180S
Amount2 µL ReagentNEBNext FFPE DNA Repair Mix - 96 rxnsNew England BiolabsCatalog #M6630L
Amount3.5 µL ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
Amount3 µL ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646

Keep master mix on ice
Add Amount12 µL 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 TemperatureRoom temperature for Duration00:10:00

10m
Incubation
Incubate samples in dry bath at Temperature65 °C for Duration00:10:00

10m
Incubation
Add 4 volumes of ReagentZymo DNA MagBinding BufferZymo ResearchCatalog #D4012-1-50 to each sample and mix well by flicking and inverting
Note
Example for calculating 4 volumes: If input is 50 µL gDNA, add 200 µL DNA MagBinding Buffer


Spin samples down briefly and add Amount20 µL ReagentZymo MagBinding BeadsZymo ResearchCatalog #D4100-5-2
Note
MagBinding Beads settle quickly, so ensure beads are kept in suspension while dispensing by vortexing beads each time before they are added to a sample


Mix samples on rotating mixer at a low speed at TemperatureRoom temperature for Duration01:30:00

1h 30m
Mix
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 Amount500 µL ReagentZymo DNA Wash BufferZymo ResearchCatalog #D4003-2-24 and then remove from magnetic stand, and mix well by flicking and inverting

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 Amount500 µL ReagentZymo DNA Wash BufferZymo ResearchCatalog #D4003-2-24 and then remove from magnetic stand, and mix well by flicking and inverting
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 Duration00:10:00

Note
MagBinding Beads utilize a different chemistry than SPRI beads (e.g., AMPure XP beads) so there is not the same risk of over-drying. It is important for optimal elution that the residual buffer is completely removed/evaporated from the beads

10m
Add Amount52 µL ReagentZymo DNA Elution BufferZymo ResearchCatalog #D3004-4-10

Manually agitate samples for Duration00:10:00 to Duration00:20:00 by gently flicking/inverting (and occasionally spinning dow to recollect liquids)
Note
This volume is too small to be able to use most rotator mixers effectively, so manually agitation is necessary


30m
Mix
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 Amount52 µL of eluate (containing repaired & end-prepped DNA) to a new clean 1.5 mL microcentrifuge tube

Use Amount1 µL of final elution to quantify via Qubit assay

Use Amount1 µL of final elution to assess fragment size distribution via TapeStation

Note
The sequencing adaptors ligated in the next section will affect the validity of TapeStation runs, so assessing the fragment distribution at this stage (i.e., after DNA repair and end preparation) is crucial for being able to estimate the molarity of your final library

Optional
Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing & NEBNext Companion Kits | ~3 hr + overnight incubation
Part 4: Adaptor ligation and clean up | ONT Ligation Sequencing & NEBNext Companion Kits | ~3 hr + overnight incubation
1h
1h
Set dry bath to Temperature37 °C

Remove ReagentAMPure XP BeadsBeckman CoulterCatalog #A63880 from storage at Temperature4 °C and allow them to come to TemperatureRoom temperature

Spin down ReagentLigation Adaptor (LA)Oxford Nanopore Technologies andReagentQuick T4 DNA LigaseNew England BiolabsCatalog #E7180S and place on ice

Thaw ReagentONT Ligation Buffer (LNB)Oxford Nanopore Technologies at TemperatureRoom temperature , spin down, and mix by pipetting. Place on ice immediately after thawing and mixing

Thaw ReagentElution Buffer (EB)Oxford Nanopore Technologies at TemperatureRoom temperature , vortex to mix, spin down, and place on ice
Thaw one tube each of ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies and ReagentLong Fragment Buffer (LFB)Oxford Nanopore Technologies at TemperatureRoom 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:
Amount25 µL ReagentLigation Adaptor (LA)Oxford Nanopore Technologies
Amount10 µL ReagentQuick T4 DNA LigaseNew England BiolabsCatalog #E7180S
Amount5 µL ReagentLigation Adaptor (LA)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:
Amount125 µL ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies
Amount375 µL ReagentLong Fragment Buffer (LFB)Oxford Nanopore Technologies

Note
For samples of sufficiently high input concentration where read length can be prioritized over gDNA retention, you may wish to instead use 1:5 SFB:LFB (i.e., 16.66 µL SBF: 88.34 µL LFB) or untitrated LFB, only

Keep titrated wash mix on ice after vortexing
Pipette Amount40 µL 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 Duration00:15:00 at TemperatureRoom temperature
Note
If you have omitted the bead-based purification steps from the second half of Part 3, do not incubate the reaction for longer than Duration00:10:00



15m
Incubation
Resuspend ReagentAMPure XP BeadsBeckman CoulterCatalog #A63880 by vortexing and add 0.4X volume resuspended beads to each sample, then flick to mix
Note
AMPure XP Beads settle quickly, so ensure beads are kept in suspension while dispensing by vortexing beads each time before they are added to a sample

Note
Example for calculating 0.4X volume: If input is 89 µL (after adding master mix), add 35.6 µL AMPure XP Beads

Mix on a rotator mixer at a low speed for Duration01:00:00 at TemperatureRoom temperature

1h
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 Amount250 µL 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 Amount250 µL 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 Duration00:00:30 to Duration00:02:00

Note
Do not allow the pellet of beads to dry to the point of cracking! Over-drying beads will result in reduced yields

2m 30s
Remove the tube from the magnetic stand and resuspend the beads in Amount15 µL ReagentElution Buffer (EB)Oxford Nanopore Technologies
Briefly spin down and incubate in dry bath at Temperature37 °C for Duration02:00:00 . During incubation, flick tube every Duration00:20:00 to agitate tissues, then briefly spin down to recollect liquids and replace tube in dry bath
Note
For HMW & uHMW gDNA, incubation at Temperature37 °C for longer times can improve the recovery of long fragments



2h 20m
Incubation
Incubate on the bench top at TemperatureRoom temperature overnight

Incubation
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 Amount15 µL of eluate (containing the prepared library) to a new clean 1.5 mL microcentrifuge tube

Use Amount1 µL of final elution to quantify library via Qubit analysis
Note
Note: For same-day or near-future sequencing, store the prepared library on ice or at Temperature4 °C until ready to be loaded onto a flow cell. Otherwise, store libraries at Temperature-20 °C