Sep 08, 2021
FindingNemo Library 3: KrazyStarFish (KSF)
- 1University of British Columbia;
- 2University of Nottingham;
- 3University of Birmingham
Protocol Citation: John Tyson, Inswasti Cahyani, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose 2021. FindingNemo Library 3: KrazyStarFish (KSF). protocols.io https://dx.doi.org/10.17504/protocols.io.bxv5pn86
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: September 01, 2021
Last Modified: September 08, 2021
Protocol Integer ID: 52893
Keywords: ultra-long sequencing, cohex, glass bead, nanopore, MinION, UHMW DNA, Monarch, Circulomics, phenol, SDS, CTAB, GM12878, Whatman, PromethION, Nanobind
Abstract
This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit.
We named this protocol KrazyStarFish (KSF). It offers a different approach to UL library prep, by using filter paper shaped as a starfish at the DNA precipitation/clean-up step. It can consistently produced N50 > 100 kb with the right transposase to DNA ratio.
This protocol was developed by John Tyson at UBC, Vancouver.
Guidelines
Acknowledgements
The FindingNemo protocol series was developed by Inswasti Cahyani for the Long Read Club with significant contributions from John Tyson and Nadine Holmes, also Josh Quick, and continuous discussion and support of Matt Loose and Nick Loman. We would also like to thank Giron Koetsier (NEB) and Kelvin Liu (Circulomics) for lending their expertise and advance product trials.
Please follow on Twitter for latest updates and results:
@NininUoN
@mattloose
Materials
Chemicals/Compounds
- Tris-HCl pH 8.0Thermo ScientificCatalog #J22638-AE
- Ethanol AbsoluteHoneywellCatalog #32221-2.5L
- Isopropanol AbsoluteFisher ScientificCatalog #P/7500/15
- Nuclease-free WaterThermofisherCatalog #AM9920
- NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
- Magnesium ChlorideFisher ScientificCatalog #AC223210010
- Triton X-100Sigma AldrichCatalog #T8787-50ML
- GlycerolBio Basic Inc.Catalog #GB0232.SIZE.500ml
Made-up Buffer
4x MuA Buffer
- 100 mM Tris-HCl pH 8.0
- 40 mM MgCl2
- 440 mM NaCl
- 0.2% TritonX-100
- 40% Glycerol
Kits
- Rapid Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-RAD004
Disposables
- DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
- DNA LoBind 2.0ml PCR Clean Eppendorf TubesEppendorfCatalog #0030 108.078
- 8-strip PCR Tubes with CapsAxygenCatalog #14-222-251
- Whatman Filter Paper No.3Catalog #SKU1153211
Note
KrazyStarFish (KSF) disk:
6 mm star-shaped (or round-shaped) disk punched out of Whatman #3 cellulose filter paper
- Wide-bore (or cut off) P1000 and P200 tips
Safety warnings
When handling phenol always wear PPE, keep a solution of 50% (w/v) PEG-400 nearby to treat the burn in the case of accidental splashes.
Before start
Things to observe at all times:
- Excessive and vigorous pipetting and vortexing should be avoided as these may shear the DNA.
- Make up buffers with nuclease-free water to avoid introducing nucleases to solutions.
- Avoid unnecessary heating and freezing; isolated DNA should be stable for storage in the fridge for months
Pre Library Prep
Pre Library Prep
This library prep is based on the rapid kit (SQK-RAD004) and using a home-made MuA buffer (see Materials).
Input DNA is based on its concentration/amount without requiring prior knowledge on input cell number.
Transposase Reaction
Transposase Reaction
In a 1.5 ml tube (labelled as DNA), gently mix 75 μl DNA (~100 ng/μl) with 25 μl 4x MuA buffer.
Note
Standard input DNA is 7.5 μg and can be for three loadings on MinION.
In another 1.5 ml tube (labelled as MuA), mix 25 μl 4x MuA buffer with 74 μl water and 0.1-1 μl FRA (depending on N50 targeted).
Note
We consistently obtained N50 >= 100 kb using a ratio of:
0.6 μl FRA per 10 μg human genome DNA
Add/mix the 100 μl content of the MuA tube into the DNA containing tube, pipetting slowly with a wide-bore P200 tip and moving the tip constantly.
Make 100 μl aliquots into PCR tubes and treat at 30oC for 1 min, 80oC for 1 min and then cool to room temperature.
30 °C 00:01:00
80 °C 00:01:00
Room temperature cool-off
DNA Precipitation
DNA Precipitation
Pool reactions into a single tube and remove ~190 μl into a fresh 1.5 ml tube.
Add 12 μl of 5 M NaCl and mix gently (by flicking or pipetting with a wide-bore P200 tip).
Add a “kRAZYsTARFISH” filter (see Materials) to the tube so it is submerged.
Add 142 μl Isopropanol to the tube.
Mix contents gently by inversion 20-30 times, allowing UHMW DNA to collect and condense onto the filter.
Pulse-spin the tube and remove the liquid by passing pipette tip past the filter.
Note
If the filter sticks to the side of the tip during this process gently place it back onto the side of the tube.
Wash the filter by addition of 500 μl 70% ethanol and gently invert the tube a few times.
Pulse-spin the tube and remove the liquid leaving the filter behind in the tube.
Repeat the 70% ethanol wash (step 12) once.
Pulse-spin the tube and remove any residual liquid leaving the filter behind and allow to air dry for a couple of minutes.
Transfer the filter to a 2 ml tube by tipping.
DNA Elution
DNA Elution
Add 125 μl of EB buffer, covering the filter, and allow tagged DNA to resuspend for 20-30 mins at 37oC with occasional gentle mixing (flicking or pipetting with a wide-bore P200 tip).
37 °C 00:30:00 max
Note
We have found efficiency of resuspension to be very good from the cellulose filter, but some gains may be had if library is left longer when using larger amounts of UHMW DNA as input.
Quantify as per section "UHMW DNA QC".
Store sample at 4oC until ready to proceed to adapter (RAP) addition followed by sequencing.
This will be sufficient for about 3 sequencing runs/reloads.
4 °C for storage
Adapter Ligation
Adapter Ligation
Remove 37.0 μl of tagged DNA library into a fresh 1.5 ml tube.
Add 37.5 μl SQB buffer, mix gently (by flicking or pipetting with a wide-bore P200 tip).
Add 0.5 μl RAP, mix gently and incubate at room temperature for 30 minutes.
Room temperature 00:30:00
Continue to Section "Flowcell Priming & Library Loading", or an optional "Nemo" clean-up step.
Note
Adding the Nemo clean-up step will remove most free adapters. In our experience testing this, the yield/occupancy was only slightly improved as this parameter depends more on the optimum transposase reaction. Using the Nemo clean-up may improve non-optimal transposase-cut library.