TITLE: Hybridization-capture for nanopore sequencing
AUTHORS: Isac Lee, Rachael Workman, Josh Zhiyong Wang, Winston Timp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Large-scale genomic anomalies – structural variations (SVs) – are pervasive in cancer. Due to the scale of the SVs and the repetitive nature of the sequences usually flanking them, they are difficult to measure with conventional short-read sequencing. The long reads possible with nanopore sequencing provide an alternative to advance the understanding of SVs. </div><div class = "text-block">In this application note, we applied SureSelectXT to nanopore long read sequencing, enriching for CDKN2A and SMAD4 tumor suppressor genes, to improve the depth and variant calling accuracy of nanopore sequencing.</div><div class = "text-block">This application note focuses on optimizing the SureSelectXT protocol to long-read sequencing and using open-source softwares nanopolish and sniffles to improve the base calling accuracy and detect single nucleotide variants (SNVs) and structural variants (SVs), demonstrating the utility of SureSelect system on third-generation long-read sequencing platforms.</div></div>

[STEPS]
?. [Post adapter-ligation PCR]
Split 40 ul eluate to 2 x 20 μL
?. [Shearing with Bioruptor Pico]
Shear 3-5 μg DNA in 100 μL to ~2kb using the following settings on Diagenode bioruptor pico : 5 cycles, 4 sec on / 30 sec off
?. [End repair and dA-tailing]
Split into two reactions to maximize the efficacy of the reactions.
?. [End repair and dA-tailing]
Mix the following components in a sterile, nuclease-free tube:  AB1Reagent
Volume
2Fragmented DNA
50 μL
3Nuclease-free water
5.5 μL
4NEBNext End Repair Reaction Buffer (10X)
6.5 μL
5NEBNext End Prep Enzyme Mix
3 μL
6Total
65 μL
AB1Reagent
Volume
2Fragmented DNA
50 μL
3Nuclease-free water
5.5 μL
4NEBNext End Repair Reaction Buffer (10X)
6.5 μL
5NEBNext End Prep Enzyme Mix
3 μL
6Total
65 μL
?. [End repair and dA-tailing]
Combine two reactions together in preparation for AMPure XP cleanup.
?. [End repair and dA-tailing]
Incubate in thermocycler :30 minutes at 20°C30 minutes at 65°Chold at 4°C
?. [AMPure XP Cleanup]
Add 65 μL of AMPure beads (1X v/v ratio) at room temperature.
?. [AMPure XP Cleanup]
Incubate for 5 minutes at room temperature.
?. [AMPure XP Cleanup]
Spin down briefly on a minifuge and place on a magnetic rack until beads separate out of the solution.
?. [AMPure XP Cleanup]
Carefully remove the supernatant, making sure not to remove the beads.
?. [AMPure XP Cleanup]
Keeping the tube on the magnetic rack, add 200 μL of 70 % ethanol in nuclease free water, being careful not to disturb the bead pellet.
?. [AMPure XP Cleanup]
Incubate for at least 30 seconds at room temperature
?. [AMPure XP Cleanup]
Repeat the wash steps (steps 9-11) for a total of two washes, and remove the supernatant (step 9).
?. [AMPure XP Cleanup]
Spin down on a minifuge to pellet the residual ethanol, place on the magnetic rack, and remove the residual ethanol using a P10 pipet.
?. [AMPure XP Cleanup]
Resuspend in 27 μL of nuclease-free water (25 + 2 to account for transfer loss.)
?. [AMPure XP Cleanup]
Incubate for 10 minutes at room temperature
?. [AMPure XP Cleanup]
Spin down briefly on a minifuge and place on a magnetic rack until beads separate out of the solution.
?. [AMPure XP Cleanup]
Retain the supernatant, which now contains the eluted DNA.
?. [Adaptor Ligation]
Add all reagents together as follows:  AB1Reagent
Volume
2Nuclease-free water
31 μL
3T4 DNA ligase buffer
20 μL
4Adapter Oligo mix
6 μL
5T4 DNA ligase
3 μL
6Template DNA
25 μL
7Total
85 μL
AB1Reagent
Volume
2Nuclease-free water
31 μL
3T4 DNA ligase buffer
20 μL
4Adapter Oligo mix
6 μL
5T4 DNA ligase
3 μL
6Template DNA
25 μL
7Total
85 μL
?. [Adaptor Ligation]
Carefully mix the reagents by pipetting up and down.
?. [Adaptor Ligation]
Briefly spin down in a microfuge.
?. [Adaptor Ligation]
Incubate in thermocycler, 20C for 15 minutes, then hold at 4C.
?. [Adaptor Ligation]
You can pause at this step and store the library at -20C
?. [Adaptor Ligation]
Purify the adapted DNA with 1.8X Ampure XP (v/v), and elute in 32 μL nuclease-free water.Refer to Ampure XP Cleanup step.
?. [Bluepippin (Sage Science) size selection]
Use 0.75% cassette high-pass for size selection protocol. (estimated time 45 minutes)
?. [Bluepippin (Sage Science) size selection]
Let it sit ~45 min after elution for best recovery.
?. [Post adapter-ligation PCR]
Add all reagents together as follows for two reactions:  AB1Nuclease-free water
2.5 μL
2NEB LongAmp Master Mix
25 μL
3SureSelect Primer (brown cap)
1.25 μL
4SureSelect R Primer (clear cap in box 2)
1.25. μL
5DNA library
20 μL
6Total
50 μL
AB1Nuclease-free water
2.5 μL
2NEB LongAmp Master Mix
25 μL
3SureSelect Primer (brown cap)
1.25 μL
4SureSelect R Primer (clear cap in box 2)
1.25. μL
5DNA library
20 μL
6Total
50 μL
?. [Post adapter-ligation PCR]
Carefully mix the reagents by inverting a few times.
?. [Post adapter-ligation PCR]
Briefly spin the reagents down on a minifuge.
?. [Post adapter-ligation PCR]
Amplify using the following cycling conditions:  ABCD1Segment Number
Number of cycles
Temperature
Time
21
1
94
30 sec
32
12
94
20 sec
45530 sec56512:30 min (50 sec/1 kb)63
1
65
10 min
74
1
4
Hold
ABCD1Segment Number
Number of cycles
Temperature
Time
21
1
94
30 sec
32
12
94
20 sec
45530 sec56512:30 min (50 sec/1 kb)63
1
65
10 min
74
1
4
Hold
?. [Post adapter-ligation PCR]
Purify the DNA with 1X Ampure XP (v/v), and elute in 15 μL nuclease-free water.Refer to Ampure XP Cleanup step.
?. [Hybridization]
Mix together these reagents to make Hyb buffer, volumes given are for one reaction:  AB1Reagent
Volume
2SureSelect Hyb 1
6.63 μL
3SS Hyb 2
0.27 μL
4SS Hyb 3
2.65 μL
5SS Hyb 4
3.45 μL
6Total
13 μL/reaction
AB1Reagent
Volume
2SureSelect Hyb 1
6.63 μL
3SS Hyb 2
0.27 μL
4SS Hyb 3
2.65 μL
5SS Hyb 4
3.45 μL
6Total
13 μL/reaction
?. [Hybridization]
Make SureSelect Block Mix.  AB1Reagent
Volume
2Indexing Block #1
2. 5 μL
3Block #2
2.5&nbsp; μL
4Indexing Blcok #3
0.6 μL
5Total
5.6 μL/reaction
AB1Reagent
Volume
2Indexing Block #1
2. 5 μL
3Block #2
2.5&nbsp; μL
4Indexing Blcok #3
0.6 μL
5Total
5.6 μL/reaction
?. [Hybridization]
Prepare RNAse block solution by diluting the RNAse block 1:10 with nuclease free water. (7 μL needed per reaction)
?. [Hybridization]
In a strip tube, add 3.4 μL gDNA to 5.6 ul SureSelect block mix
?. [Hybridization]
Mix by pipetting, and denature the DNA on a thermocycler by incubating at 95C for 5 minutes, followed by incubationg at 65C for at least 5 minutes
?. [Hybridization]
Meanwhile, make the capture library mix :  AB1Reagent
Volume
2Hyb buffer mix
13 μL
3RNAse block dilution
5 μL
4Capture library
2 μL
5Total
20 μL/reaction
AB1Reagent
Volume
2Hyb buffer mix
13 μL
3RNAse block dilution
5 μL
4Capture library
2 μL
5Total
20 μL/reaction
?. [Hybridization]
Keep at room temperature briefly, and add to the denatured DNA while the DNA is still on the thermocycler
?. [Hybridization]
Incubate for 16 or 24 hours at 65C with heated lid at 105C.
?. [Streptavidin beads preparation]
Prewarm SureSelectXT Wash buffer #2 at 65C.
?. [Streptavidin beads preparation]
Vigorously resuspend MyOne Streptavidin T1 beads on a vortex mixer.
?. [Streptavidin beads preparation]
Add 50 μL beads (per rxn) to a 1.5 mL tube.
?. [Streptavidin beads preparation]
Add 200 μL of SureSelect Binding buffer.
?. [Streptavidin beads preparation]
Mix the beads on a vortex mixer for 5 seconds.
?. [Streptavidin beads preparation]
Put the tubes into a magnetic rack
?. [Streptavidin beads preparation]
Remove and discard the supernatant.
?. [Streptavidin beads preparation]
Repeat above for a total of 3 washes.
?. [Streptavidin beads preparation]
Resuspend the beads in 200 μL of SureSelectXT Binding buffer
?. [Capture]
Take out hybridization mixture from thermal cycler, add directly to bead solution, invert 3-5X to mix.*If less than 20 μL hyb mixture remains after incubation, be wary of suboptimal capture performance.
?. [Capture]
Incubate hybrid-bait/bead solution on tube rotator at 10 rpm top-to-bottom for 30 minutes at room temperature. If sample is not properly mixing, mix with heat using a thermomixer at 30 C 800 rpm.
?. [Capture]
Briefly spin down on a minifuge.
?. [Capture]
Place on magnetic rack, remove and discard supernatant.
?. [Capture]
Resuspend in 500 μL wash buffer I by vortexing for 5 seconds
?. [Capture]
Incubate for 15 minutes at room temperature, occasionally mixing.
?. [Capture]
Spin down briefly on a minifuge, place on magnetic rack, and remove supernatant.
?. [Capture]
Resuspend beads in 200 μL 65C prewarmed wash buffer, mix on a vortex mixer for 5 seconds to resuspend beads.
?. [Capture]
Incubate 10 minutes at 65C
?. [Capture]
Briefly spin down on a minifuge, place on magnetic rack, and discard supernatant. Repeat the wash steps for a total of 3 washes.
?. [Capture]
Remove all wash buffer
?. [Capture]
Add 30 μL nuclease free water to each sample, pipet to resuspend. Keep samples on ice until next use.
?. [Capture Library Amplification]
Assemble PCR reaction, 2 for each capture reaction :   AB1Reagent
Volume
2Nuclease-free water
5 μL
3NEB Long AMP MM
25 μL
4SureSelect ILM post-capture forward PCR primer
1 μL
5SS indexed primer
5 μL
6Bead-bound DNA
14 μL
7Total
50 μL
AB1Reagent
Volume
2Nuclease-free water
5 μL
3NEB Long AMP MM
25 μL
4SureSelect ILM post-capture forward PCR primer
1 μL
5SS indexed primer
5 μL
6Bead-bound DNA
14 μL
7Total
50 μL
?. [Capture Library Amplification]
Amplify using the following cycling conditions:  ABCD1Segment Number
Number of cycles
Temperature
Time
21
1
94
30 sec
32
14
94
15 sec
46030 sec5658:20 min63
1
65
10 min
74
1
4
Hold
ABCD1Segment Number
Number of cycles
Temperature
Time
21
1
94
30 sec
32
14
94
15 sec
46030 sec5658:20 min63
1
65
10 min
74
1
4
Hold
?. [Oxford Nanopore sequencing library preparation (Protocol for SQK-LSK108 Kit)]
Perform NEBNext Ultra II End-Repiar/dA-tailing in 60 μL.
?. [Capture Library Amplification]
Purify the DNA with 1X Ampure XP (v/v), and elute in 30 μL nuclease-free water.Refer to Ampure XP Cleanup step.
?. [Oxford Nanopore sequencing library preparation (Protocol for SQK-LSK108 Kit)]
Purify the DNA with 1X Ampure XP (v/v), and elute n 30 μL nuclease-free water.
?. [Oxford Nanopore sequencing library preparation (Protocol for SQK-LSK108 Kit)]
Ligate on adaptors using NEB Blunt/TA Ligase Master Mix.
?. [Oxford Nanopore sequencing library preparation (Protocol for SQK-LSK108 Kit)]
Purify the adptor-ligated DNA following the protocol's specifications.
?. [Oxford Nanopore sequencing library preparation (Protocol for SQK-LSK108 Kit)]
Load on ONT MinION following the protocol's specifications and sequence for up to 48 hours.