Long library preparation
In this step, long libraries are prepared and Illumina adaptors are ligated.
DNA shearing
We tested two Covaris-based options to shear DNA; Red miniTUBE (this protocol) and gTUBE for longer fragments (>5 kb).
1. Red miniTUBE on Covaris S2.
Shear 2-4 μg of gDNA with the following settings (5 kb):
• Intensity – 1
• Duty Cycle – 20%
• Cycles per Burst – 1000
• Treatment time – 600 s
• Temperature – 20°C
• Water level – 15
• Sample volume – 200 μl
Check whether DNA was sheared to the desired size on 0.8% agarose gel with a suitable DNA marker.
2. Purification and size selection on AMPure XP beads
This purification step includes also first size selection with 0.4X AMPure XP beads.
• To the sheared DNA (190 μl) in a sterile, nuclease-free Eppendorf tube, add 76 μl (0.4X) resuspended by vortexing AMPure XP beads and mix well by pipetting up and down at least 10 times.
• Incubate for 5 minutes at room temperature.
• Spin briefly (1-2 s) and place the tube on a magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube. Be careful not to disturb the beads that contain the target DNA fragments.
• Add 200 μl of freshly prepared 80% ethanol to the tube while on the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
• Repeat wash step twice, to three washes in total.
• Briefly spin the tube (1-2 s) and put it back on the magnetic stand.
• Completely remove the residual ethanol and air dry beads for up to 10 minutes while the tube is on the magnetic stand with lid open.
• Elute the DNA target from the beads with 65 μl nuclease-free water. Mix well by pipetting up and down and put the tube on the magnetic stand until the solution is clear.
• Transfer the supernatant to a sterile, nuclease-free Eppendorf tube.
Check size-selected fraction on 0.8% agarose gel (it should contain fragments >2 kb).
Size selection is optional for longer libraries (>3 kb).
3. Enrichment library construction
To construct a DNA library for sequence capture, the NEBNext Ultra DNA Library Prep Kit for Illumina is used according to the manufacturer’s protocol, with minor modifications. The recommended input is 500 ng-1 μg of sheared and size-selected DNA. Lower amounts are acceptable, but will require some adjustments (see NEBNext protocol for details).
Note: Other kits can also be used, but check for compatibility with downstream oligo blocks and adaptors.
4. End repair
Before starting, make sure that DTT in End Repair Reaction buffer is fully dissolved.
• Mix the following components in a sterile, nuclease-free Eppendorf tube:
End Prep Enzyme Mix – 3.0 μl
End Repair Reaction Buffer (10X) – 6.5 μl
Fragmented DNA – 55.5 μl
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Total volume – 65 μl
• Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
• Incubate as below:
30 minutes at 20°C
30 minutes at 65°C
5 minutes on ice.
5. Adaptor ligation
• Add the following components directly to the End Prep reaction mixture and mix well:
Blunt/TA Ligase Master Mix – 15 μl
NEBNext Adaptor for Illumina – 2.5 μl
Ligation Enhancer – 1 μl
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Total volume – 83.5 μl
• Mix by pipetting, followed by a quick spin to collect all liquid from the sides of the tube.
• Incubate at 20°C for 30 minutes.
• Add 3 μl of USER enzyme to the ligation mixture above.
• Mix well and incubate at 37°C for 20 minutes.
6. Library purification
For this step, use ~0.5X AMPure beads as there is no need to perform a more stringent size selection step here.
• Add 43 μl resuspended by vortexing AMPure XP beads to the ligation reaction. Mix well by pipetting up and down at least 10 times.
• Incubate for 5 minutes at room temperature.
• Spin briefly (1-2 s) and place the tube on a magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube. Be careful not to disturb the beads that contain the target DNA fragments.
• Add 200 μl of freshly prepared 80% ethanol to the tube while on the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
• Repeat wash step twice, to three washes in total.
• Briefly spin the tube (1-2 s) and put it back on the magnetic stand.
• Completely remove the residual ethanol and air dry beads for up to 10 minutes while the tube is on the magnetic stand with lid open.
• Elute the DNA target from the beads with 25 μl nuclease-free water. Mix well by pipetting up and down and put the tube on the magnetic stand until the solution is clear.
• Transfer the supernatant to a sterile, nuclease-free Eppendorf tube.
7. PCR amplification
Mix the following components in a sterile, nuclease-free PCR tube:
Adaptor Ligated DNA Fragments – 10 μl*
2X KAPA HiFi HotStart ReadyMix – 25 μl
Index Primer – 1 μl
Universal PCR Primer – 1 μl
Water – 13 μl
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Total volume – 50 μl
PCR cycling conditions:
Initial denaturation: 98°C, 4 minutes
8-15 cycles with the following settings:
Denaturation: 98°C, 30 seconds
Annealing: 65°C, 30 seconds
Extension: 72°C, 4 minutes
Final extension 72°C, 10 minutes.
==*== The amount of the library used for amplification may require optimization for different libraries.
• First perform 8 cycles and run 5 μl of the reaction on 0.8% agarose gel next to 1 μl of unamplified library (as a control of input DNA). In case there is not enough DNA, put the reaction back to thermocycler for additional 2-5 cycles.
8. Library size selection and purification
• Transfer the PCR reaction (45 μl) to a sterile, nuclease-free Eppendorf tube, add 18 μl (0.4X) of resuspended by vortexing AMPure XP beads and mix well by pipetting up and down at least 10 times.
• Incubate for 5 minutes at room temperature.
• Spin briefly (1-2 s) and place the tube on a magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube. Be careful not to disturb the beads that contain the target DNA fragments.
• Add 200 μl of freshly prepared 80% ethanol to the tube while on the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
• Repeat wash step twice, to three washes in total.
• Briefly spin the tube (1-2 s) and put it back on the magnetic stand.
• Completely remove the residual ethanol and air dry beads for up to 10 minutes while the tube is on the magnetic stand with lid open.
• Elute the DNA target from the beads with 20 μl nuclease-free water. Mix well by pipetting up and down and put the tube on the magnetic stand until the solution is clear.
• Transfer the supernatant to a sterile, nuclease-free Eppendorf tube.
Check size-selected fraction on 0.8% agarose gel.
9. Hybridization
The enrichment of target DNA fragments is achieved through hybridization of the PCR amplified genomic libraries (Section 2) with complementary RNA (cRNA) baits (bait library). The following protocol is based on the MYbaits protocol and kit (Agilent SureSelect can be easily adapted as well). The high capture efficiency observed over time allowed to perform the hybridization in a half of reaction volume.
Note: Hybridization setup is based on MYbaits protocol v3. For more information, see MYbaits protocol: www.mycroarray.com/pdf/MYbaits-manual-v3.pdf
Before starting, equilibrate HYB #4 tube at room temperature to fully dissolve SDS that may have precipitated during storage at 4°C.
10. Library Master Mix Preparation
Note 1: The recommended input for half a reaction volume is 250-350 ng of library DNA in 3.5 μl. It may be necessary to concentrate the genomic library before further processing by reducing the volume using a SpeedVac.
Note 2: Although Block #1 and Block #2 from MYbaits kit can be used for plant based enrichment, enrichment efficiency is higher when these blockers are replaced by Roche SeqCap EZ Developer Reagent.
Note 3: Make sure that Block #3 matches adapters used for long library preparation.
In a sterile, nuclease-free PCR tube, mix:
SeqCAP – 2.5 μl
Block #3 – 0.3 μl
Long Illumina library from step 2.2.5 (250-500 ng) – 3.5 μl
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Total volume – 6.3 μl
Transfer the tube containing the Library Master Mix to the thermocycler and incubate at 95°C for 5 minutes and then hold at 65°C, with the ‘heated lid’ option enabled. While Library Master Mix is incubated in the thermocycler, proceed to Hybridization Master Mix preparation.
11. Hybridization Master Mix Preparation
In a sterile, nuclease-free PCR tube, mix:
Hyb #1 – 4.5 µl
Hyb #2 – 0.25 µl
Hyb #3 – 1.75 µl
Hyb #4 – 0.25 µl
RNase Block – 0.5µl
Baits – 2.5 µl
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Total volume – 9.25 μl
• Mix the components by vortexing, followed by a quick spin to collect all liquid from the sides of the tube.
• Transfer the tube containing the Hybridization Master Mix to the thermocycler and incubate at 65°C for 5 minutes.
• While keeping the tube at 65°C, transfer 9 μl of Hybridization Master Mix to the Library Master Mix and mix by pipetting.
• Hybridize solution at 65°C for 16-24 hours.
12. Recovery of captured targets
Before starting, preheat Wash Buffer 2 to 65°C for at least 1 hour, then prepare Wash Buffer 2.2 as follows:
• In a 50 ml tube, combine 400 μl HYB #4, 39.6 ml nuclease-free water and 10 ml Wash Buffer 2.
• Heat the Wash Buffer 2.2 to 65°C for at least 45 minutes before use.
The prepared volume of Wash Buffer 2.2 is sufficient for washing 33 samples. It can be stored at 4°C for up to 6 weeks.
13. Capture and washing
• Transfer 20 μl of MyOne Streptavidin C1 magnetic beads to a sterile, nuclease-free Eppendorf tube.
• Place the tube on a magnetic stand to separate beads from supernatant. After the solution is clear, carefully remove and discard the supernatant.
• Add 200 μl Binding Buffer to wash the beads. Vortex the tube for 5-10 seconds, place on the magnetic stand for 2 minutes, and then carefully remove and discard the supernatant.
• Repeat wash step twice for a total of three washes.
• Resuspend the beads in 35 μl Binding Buffer and incubate at 65°C for 2 minutes.
• Transfer the hybridization solution to the Binding Buffer/Beads and incubate 45min at 65°C, mixing solution every 5-10 minutes.
• Pellet the beads on the magnetic stand for 2 minutes and carefully remove and discard the supernatant.
• Add 500 μl of the Wash Buffer 2.2 from 65°C to the beads and mix by pipetting. Incubate for 10 minutes at 65°C in a thermal block. Flick the tube occasionally to resuspend the beads. Pellet the beads on the magnetic stand for 2 minutes and carefully remove and discard the supernatant.
• Repeat washing step twice for a total of three 65°C washes. After the third wash, make sure all additional buffer is removed by giving the tube a quick spin after the supernatant has been removed, and re-pelleting the beads with the magnetic stand.
• Resuspend the beads in 20 μl molecular biology grade water.
14. Amplification of the captured library
This step consists of amplifying the captured DNA while it is still attached to the streptavidin beads. It is important to limit the number of cycles to get just enough material for sequencing while minimizing PCR amplification bias. For this step, KAPA HiFi DNA Polymerase was used which compares favourably to other available DNA polymerases.
• Prepare a 50 μl PCR reaction as follows on ice in a nuclease-free PCR tube, and mix by pipetting:
Captured Library – 1 μl*
2X KAPA HiFi HotStart ReadyMix – 25 μl
P5 primer (10 μM) – 1.5 μl
P7 primer (10 μM) – 1.5 μl
Water – 22.5 μl
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Total volume – 50 μl
• PCR cycling conditions:
Initial denaturation: 98°C, 4 minutes
25 cycles with the following settings:
Denaturation: 98°C, 30 seconds
Annealing: 65°C, 30 seconds
Extension: 72°C, 4 minutes
Final extension: 72°C, 10 minutes.
==*== The amount of the enriched library (from step 3.2.1) used for amplification may require optimization for different libraries.
• Check 5 μl of PCR product on the 0.8% agarose gel – usually, 25 cycles yield 1-2 μg of the amplified enriched library; therefore, 5 μl should be well visible on gel. If amplification is weak, perform 2-5 additional cycles.
• It is highly recommended to perform enrichment efficiency check using qPCR. Primer should be designed in a few genes used for enrichment. Difference between enriched and non enriched samples will oscillate between 8-11 cycles.