TITLE: FindingNemo Library 3: KrazyStarFish (KSF)
AUTHORS: John Tyson, Inswasti Cahyani, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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, </span><span style = "font-style:italic;">i.e., </span><span style = "font-weight:bold;">SQK-RAD004</span><span>, a transposase based adapter ligation kit.</span></div><br/><div class = "text-block"><span>We named this protocol </span><span style = "font-weight:bold;">KrazyStarFish (KSF)</span><span>. 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.</span></div><br/><div class = "text-block">This protocol was developed by John Tyson at UBC, Vancouver.</div></div>

[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.
?. [DNA Tagmentation]
In a 1.5 ml tube (labelled as DNA), gently mix 75 μl DNA (~100 ng/μl) with 25 μl 4x MuA buffer.
Standard input DNA is 7.5 μg and can be for three loadings on MinION.
?. [DNA Tagmentation]
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).
We consistently obtained N50 >= 100 kb using a ratio of:0.6 μl FRA per 10 μg DNA
?. [DNA Tagmentation]
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.
?. [DNA Tagmentation]
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
80 °C
[cool-off]
?. [DNA Tagmentation]
Pool reactions into a single tube and remove 190 μl into a fresh 1.5 ml tube.
?. [DNA Precipitation]
Add 12 μl of 5M NaCl and mix gently (by flicking or pipetting with a wide-bore P200 tip).
?. [DNA Precipitation]
Add a “kRAZYsTARFISH” filter (see Materials) to the tube so it is submerged.
?. [DNA Precipitation]
Add 142 μl Isopropanol to the tube.
?. [DNA Precipitation]
Mix contents gently by inversion 20-30 times, allowing UHMW DNA to collect and condense onto the filter.
?. [DNA Precipitation]
Pulse-spin the tube and remove the liquid by passing pipette tip past the filter.
If the filter sticks to the side of the tip during this process gently place it back onto the side of the tube.
?. [DNA Precipitation]
Wash the filter by addition of 500 μl 70% ethanol and gently invert the tube a few times.
?. [DNA Precipitation]
Pulse-spin the tube and remove the liquid leaving the filter behind in the tube.
?. [DNA Precipitation]
Repeat the 70% ethanol wash (steps 48-49) once.
?. [DNA Precipitation]
Pulse-spin the tube and remove any residual liquid leaving the filter behind and allow to air dry for a couple of minutes.
?. [DNA Precipitation]
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
[max]
We have found efficiency of resuspension to be very good off the cellulose filter, but some gains may be had if library is left longer when using larger amounts of UHMW DNA as input.
?. [DNA Elution]
Quantify as per section "UHMW DNA QC".
?. [DNA Elution]
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.
0 °C
[for storage]
?. [Adapter Ligation]
Remove 37.0 μl of tagged DNA library into a fresh 1.5 ml tube.
?. [Adapter Ligation]
Add 37.5 μl SQB buffer, mix gently (by flicking or pipetting with a wide-bore P200 tip).
?. [Adapter Ligation]
Add 0.5 μl RAP, mix gently and incubate at room temperature for 30 minutes.
0 Room temperature
?. [Adapter Ligation]
Continue to Section "Flowcell Priming & Library Loading", or an optional "NEMO" clean-up step.
Adding Nemo clean-up step will remove most free adapters. In our limited experience testing this, the yield/occupancy was only slightly improved as this parameter depends more on the optimum 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.
Standard input DNA is 7.5 μg and can be for three loadings on MinION.
?. [Transposase Reaction]
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).
We consistently obtained N50 >= 100 kb using a ratio of:0.6 μl FRA per 10 μg human genome DNA
?. [Transposase Reaction]
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.
?. [Transposase Reaction]
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
80 °C
[cool-off]
?. [DNA Precipitation]
Pool reactions into a single tube and remove ~190 μl into a fresh 1.5 ml tube.
?. [DNA Precipitation]
Add 12 μl of 5 M NaCl and mix gently (by flicking or pipetting with a wide-bore P200 tip).
?. [DNA Precipitation]
Add a “kRAZYsTARFISH” filter (see Materials) to the tube so it is submerged.
?. [DNA Precipitation]
Add 142 μl Isopropanol to the tube.
?. [DNA Precipitation]
Mix contents gently by inversion 20-30 times, allowing UHMW DNA to collect and condense onto the filter.
?. [DNA Precipitation]
Pulse-spin the tube and remove the liquid by passing pipette tip past the filter.
If the filter sticks to the side of the tip during this process gently place it back onto the side of the tube.
?. [DNA Precipitation]
Wash the filter by addition of 500 μl 70% ethanol and gently invert the tube a few times.
?. [DNA Precipitation]
Pulse-spin the tube and remove the liquid leaving the filter behind in the tube.
?. [DNA Precipitation]
Repeat the 70% ethanol wash (step 12) once.
?. [DNA Precipitation]
Pulse-spin the tube and remove any residual liquid leaving the filter behind and allow to air dry for a couple of minutes.
?. [DNA Precipitation]
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
[max]
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.
?. [DNA Elution]
Quantify as per section "UHMW DNA QC".
?. [DNA Elution]
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.
[for storage]
?. [Adapter Ligation]
Remove 37.0 μl of tagged DNA library into a fresh 1.5 ml tube.
?. [Adapter Ligation]
Add 37.5 μl SQB buffer, mix gently (by flicking or pipetting with a wide-bore P200 tip).
?. [Adapter Ligation]
Add 0.5 μl RAP, mix gently and incubate at room temperature for 30 minutes.
0 Room temperature
?. [Adapter Ligation]
Continue to Section "Flowcell Priming & Library Loading", or an optional "Nemo" clean-up step.
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.