TITLE: FindingNemo Library 2: Modified RAD004
AUTHORS: Inswasti Cahyani, John Tyson, 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>This </span><span style = "font-weight:bold;">Modified RAD004</span><span> protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA, for when ULK001 is not accessible/available. Transposase-based reaction is done in a large volume of up to 1 ml.</span></div><br/><div class = "text-block">The working principle of the RAD004 kit is shown in the diagram below:</div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/echs7t5n.jpg" /></div><br/></div>

[STEPS]
?. [Library Prep Notes]
Extracted UHMW DNA is often difficult to quantify due to its viscosity. However, accurate measurement of DNA concentration is crucial for calculating optimum ratio of the transposase enzyme to the DNA molecules. We provide a protocol section for quantifying UHMW DNA in our 'FindingNemo' protocol master file.Properly quantified DNA can then be processed for this library prep.Both cell number and DNA concentration/amount are used to calculate the amount of transposase (FRA) and adapter (RAP). We base this protocol on SQK-RAD004 but follow the SQK-ULK001 protocol for the optimum ratio of transposase amount to human genomic DNA:6 μl FRA to 6 million human cells (or around 40 μg DNA)For other species, the genome size has to be taken into account and the FRA to DNA ratio optimised, e.g., we had optimised a non-human cell line of 6.2 Gb genome at: 2.5 μl FRA to 1 million non-human cell (around 12-15 μg DNA)
?. [UL Library Prep]
The following table lists the replaced chemicals between ULK001 and RAD004.Chemical ULK001RAD004 (modified)Dilution bufferFDBMuA buffer (see Materials)TransposaseFRA*FRA^Sequencing adapterRAP-FRAP
Chemical ULK001RAD004 (modified)Dilution bufferFDBMuA buffer (see Materials)TransposaseFRA*FRA^Sequencing adapterRAP-FRAP
?. [UL Library Prep]
In a 2 ml tube, dilute UHMW DNA to a concentration of around 50 ng/μl in a total volume of 750 μl (with water or elution buffer if required).Mix well with a P1000 wide-bore tip. Cell No. (million)Approx. DNA amount (μg)Total DNA volume (μl)DNA concentration (ng/μl)Total reaction volume (μl)6>20-4075020-5010005435-2037550021
DNA concentration can still range from 20-50 ng/l to have optimum tagmentation reaction.If input DNA amount is less than 20 μg (1-3 million cells used), halve all the reaction volumes, i.e., 375 μl total DNA volume instead of 750 μl as in the table below.It is important to have as homogeneous DNA as possible at this step so the transposase can access and cut the DNA solution with an even distribution. It is OK to pipette thoroughly but gently.
Cell No. (million)Approx. DNA amount (μg)Total DNA volume (μl)DNA concentration (ng/μl)Total reaction volume (μl)6>20-4075020-5010005435-2037550021
?. [UL Library Prep]
In a 1.5 ml tube, dilute the corresponding amount of transposase (FRA) with the dilution buffer (FDB) to a total volume of 250 μl (or 125 μl if doing half-reaction). More details in the table below.Cell No. (million)Approx. DNA amount (μg)FRA (μl)4X MuA Buffer (μl)Total reaction volume (μl)6>20-4062441000552454424635-2031225002212311124
Cell No. (million)Approx. DNA amount (μg)FRA (μl)4X MuA Buffer (μl)Total reaction volume (μl)6>20-4062441000552454424635-2031225002212311124
?. [UL Library Prep]
Mix the diluted FRA by vortexing for 2-3 seconds.
?. [UL Library Prep]
Using a P1000 wide-bore tip, add the diluted FRA to the DNA sample. Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.Mix thoroughly by gentle pipetting.
on ice
?. [UL Library Prep]
Incubate the reaction as follows:
23 °C
70 °C
0 Room temperature
[at least]
It is important that the room temperature at the fragmentation step (first incubation step) does not fall below 20oC to ensure optimum reaction condition. The use of a water bath or heating block is recommended.
?. [UL Library Prep]
Add the corresponding volume of sequencing adapter (RAP) as in the table below. Cell No. (million)Approx. DNA amount (μg)FRA (μl)RAP (μl)Total reaction volume (μl)6>20-40651000554.2443.335-2032.5500221.7110.8
Cell No. (million)Approx. DNA amount (μg)FRA (μl)RAP (μl)Total reaction volume (μl)6>20-40651000554.2443.335-2032.5500221.7110.8
Use a P1000 wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed. Tube inversion can be used to aid mixing.
?. [UL Library Prep]
Incubate for 30 minutes at 23oC.
23 °C
?. [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.
?. [Library Prep Notes]
The following table lists the replaced reagents between ULK001 and RAD004.ReagentULK001RAD004 (modified)Dilution bufferFDBMuA buffer (see Materials)TransposaseFRA*FRA^Sequencing adapterRAP-FRAP
ReagentULK001RAD004 (modified)Dilution bufferFDBMuA buffer (see Materials)TransposaseFRA*FRA^Sequencing adapterRAP-FRAP
?. [Transposase Reaction]
In a 2 ml tube, dilute UHMW DNA to a concentration of around 50 ng/μl in a total volume of 750 μl (with water or elution buffer if required).Mix well with a P1000 wide-bore tip. Cell No. (million)Approx. DNA amount (μg)Total DNA volume (μl)DNA concentration (ng/μl)Total reaction volume (μl)6>20-4075020-5010005435-2037550021
DNA concentration can still range from 20-50 ng/μl to have optimum transposase reaction.If input DNA amount is less than 20 μg (1-3 million cells used), halve all the reaction volumes, i.e., 375 μl total DNA volume instead of 750 μl as in the table below.It is important to have as homogeneous DNA as possible at this step so the transposase can access and cut the DNA molecules with an even distribution. It is OK to pipette thoroughly but gently.
Cell No. (million)Approx. DNA amount (μg)Total DNA volume (μl)DNA concentration (ng/μl)Total reaction volume (μl)6>20-4075020-5010005435-2037550021
?. [Transposase Reaction]
In a 1.5 ml tube, dilute the corresponding amount of transposase (FRA) with MuA buffer to a total volume of 250 μl (or 125 μl if doing half-reaction). More details in the table below.Cell No. (million)Approx. DNA amount (μg)FRA (μl)4X MuA Buffer (μl)Total reaction volume (μl)6>20-4062441000552454424635-2031225002212311124
Cell No. (million)Approx. DNA amount (μg)FRA (μl)4X MuA Buffer (μl)Total reaction volume (μl)6>20-4062441000552454424635-2031225002212311124
?. [Transposase Reaction]
Mix the diluted FRA by vortexing for 2-3 seconds.
?. [Transposase Reaction]
Using a P1000 wide-bore tip, add the diluted FRA to the DNA sample. Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.Mix thoroughly by gentle pipetting.
on ice
?. [Transposase Reaction]
Incubate the reaction as follows:
23 °C
70 °C
0 Room temperature
[at least]
It is important that the room temperature at the fragmentation step (first incubation step) does not fall below 20oC to ensure optimum reaction condition. The use of a water bath or heating block is recommended.
?. [Adapter Ligation]
Add the corresponding volume of sequencing adapter (RAP) as in the table below. Cell No. (million)Approx. DNA amount (μg)FRA (μl)RAP (μl)Total reaction volume (μl)6>20-40651000554.2443.335-2032.5500221.7110.8
Cell No. (million)Approx. DNA amount (μg)FRA (μl)RAP (μl)Total reaction volume (μl)6>20-40651000554.2443.335-2032.5500221.7110.8
Use a P1000 wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed. Tube inversion can be used to aid mixing.
?. [Adapter Ligation]
Incubate for 30 minutes at 23oC.
23 °C