FindingNemo Library 3: KrazyStarFish (KSF)
Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose
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.
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
Steps
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
In a 1.5 ml tube (labelled as DNA), gently mix 75 μl DNA (~100 ng/μl) with 25 μl 4x MuA buffer.
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).
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 0h 1m 0s
80°C 0h 1m 0s
Room temperature
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.
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
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 0h 30m 0s
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
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 0h 30m 0s
Continue to Section "Flowcell Priming & Library Loading", or an optional " Nemo " clean-up step.
