Whole Exome Sequencing Based on Hybridization Capture Protocol

Purification

1. Let AMPure XP beads come to room temperature for at least 30 min.
2. Mix the reagent well on a vortex mixer till it appears homogenous. Add 180 μl of homogenous AMPure XP beads to a 1.5-ml LoBind tube, and add the sheared DNA library (~120 μl).
3. Mix well on a vortex mixer and incubate for 5 min at room temperature.
4. Put the tube in a magnetic stand and wait for the solution to clear (3–5 min).
5. Keep the tube in the magnetic stand. Discard the cleared solution from the tube without disturbing the beads.
6. Continue to keep the tube in the magnetic stand while you dispense 500 μl of 70% ethanol in each tube.
7. Let the tube sit for 1 min to allow any disturbed beads to settle, and then remove ethanol. Repeat this step once.
8. Dry the samples on a 37°C heat block for 5 min or until the residual ethanol completely evaporates.
9. Add 30 μl nuclease-free water, mix well on a vortex mixer, and incubate for 2 min at room temperature.
10. Place the tube in the magnetic stand and leave for 2–3 min or until the solution is clear.
11. Remove approximately 30 μl of the supernatant to a fresh 1.5-ml LoBind tube.

Assess Quality

1. Use Agilent DNA 1000 chip and reagent kit for preparing the chip with samples and ladder.
2. Load the chip into the 2100 Bioanalyzer and start the run within 5 min after preparation.
3. Check that the electropherogram shows a distribution with a peak size of ~190 nucleotides.

End Repair

1. Prepare the reaction mix on ice. Mix well on a vortex mixer. Add 71 μl of the reaction mix and 29 μl of DNA sample to each tube. Mix by pipetting.
2. Incubate tube in a thermal cycler for 30 min at 20°C. Do not use heated lid.
3. Purify the sample using 90 μl of homogenous AMPure XP beads, following the instruction. Elute in 32 μl nuclease-free water.

A-Tailing

1. Prepare the reaction mix. Add 32 μl of each DNA sample to each well or tube. Mix by pipetting.
2. Incubate in a thermal cycler for 30 min at 37°C with the lid temperature not exceeding 50°C.
3. Purify the sample using 90 μl of homogenous AMPure XP beads, following the instruction, and elute in 15 μl nuclease-free water.
4. Proceed immediately to adapter ligation step.

Adapter Ligation

1. Anneal the adapters.
2. Prepare the reaction mix.
3. Add 36 μl of the reaction mix and 14 μl of DNA sample to each tube. Mix by pipetting.
4. Incubate for 15 min at 20°C on a thermal cycler. Do not use a heated lid.
5. Purify the sample using 90 μl of homogenous AMPure XP beads, following the instruction. Elute in 52 μl nuclease-free water.

Pre-hybridization Amplification

1. Use half of the adapter-ligated fragments for amplification and save the other half at 20°C for future use, if needed.
2. Prepare the reaction mix (25 μl) as instructed in Table 5 and add 25 μl of each DNA sample to each tube
3. Mix by pipetting and run the program.
4. Purify the sample using 90 μl of homogenous AMPure XP beads and elute in 30 μl nuclease-free water.

Assess Quality and Quantity

1. Use Agilent DNA 1000 chip and reagent kit for preparing the chip with samples and ladder.
2. Load the chip into the 2100 Bioanalyzer and start the run within 5 min after preparation.
3. After the run, determine the concentration of the sample by integration under the peak.
4. Check that the electropherogram shows a distribution with a peak size of 250 ± 10% bp.

Hybridization

1. Concentrate a 500-ng aliquot of library to 3.4 μl using a vacuum concentrator at ≤45°C.
2. Mix the components to prepare block mix in a PCR tube.
3. Add 3.4 μl of 147 ng/μl prepped library with 5.6 μl of SureSelect Block Mix (Tube A).
4. Mix the components in Table 8 at room temperature to prepare the hybridization buffer (Tube B).
5. Dilute 1 μl RNase Block with 2 μl nuclease-free water.
6. In a separate tube, mix 5 μl of SureSelect capture library with 2 μl of diluted RNase Block (Tube C).
7. Place the tube "A" in thermal cycler with the following program
8. Use a heated lid on the thermal cycler at 105°C throughout.
9. Maintain the cycler at 65°C while you add 40 μl of hybridization buffer (Tube B).
10. Incubate both tubes for a minimum of 5 min at 65°C.
11. Place the capture library mix (Tube C) in cycler.
12. Incubate the samples at 65°C for 2 min.
13. Maintain the cycler at 65°C while using pipette to take 13 μl of Hybridization Buffer from the tube "B" and add it to SureSelect capture library mix tube "C."
14. Maintain the cycler at 65°C and transfer the entire content of prepped library mix in tube "A" to the hybridization solution in tube "C."
15. Seal the tube "C" carefully and incubate the hybridization mixture for 24 h at 65°C with a heated lid at.

Magnetic Beads' Preparation

1. Keep SureSelect Wash Buffer #2 at 65°C water bath.
2. Let the Dynal MyOne Streptavidin T1 (Invitrogen) beads come to room temperature for at least 30 min.
3. Vigorously resuspend on a vortex mixer and take 50 μl of magnetic beads to a 1.5-ml microfuge tube.
4. Wash the beads:
(a) Add 200 μl of SureSelect Binding buffer.
(b) Mix the beads on a vortex mixer for 5 s.
(c) Place the tube into a magnetic stand and wait till solution becomes clear (3–5 min).
(d) Carefully remove and discard the supernatant.
(e) Repeat steps (a through d) for a total of three washes.
5. Resuspend the beads in 200 μl of SureSelect Binding buffer.

Hybrid Capture Selection

1. Estimate and record the volume of hybridization that remained after 24 h incubation.
2. Add the hybridization mixture directly from the thermal cycler to the bead solution, and mix by inverting three to five times.
3. Incubate the hybrid-capture/bead solution on a Nutator or equivalent for 30 min at room temperature.
4. Make sure that the content is mixing properly.
5. Briefly spin in a centrifuge and separate the beads and buffer on a magnetic separator.
6. Keep the tube in the magnetic stand for 3–5 min.
7. Discard the cleared solution from the tube without disturbing the beads.
8. Resuspend the beads in 500 μl of SureSelect Wash Buffer #1 by mixing on a vortex mixer for 5 s.
9. Incubate the samples for 15 min at room temperature. Occasionally mix on a vortex mixer.
10. Briefly spin in a centrifuge, separate the beads and buffer on a magnetic separator, and remove the supernatant.
11. Wash the beads:
(a) Resuspend the beads in 500 μl of 65°C pre-warmed SureSelect Wash Buffer #2 and mix on a vortex mixer for 5 s to resuspend the beads.
(b) Incubate the samples for 10 min at 65°C in a heat block or equivalent. Occasionally mix on a vortex mixer.
(c) Briefly spin in a centrifuge.
(d) Separate the beads and buffer on a Dynal magnetic separator and remove the supernatant.
(e) Repeat steps (a through d) for a total of three washes. Remove all traces of wash buffer before proceeding to step 12.
12. Mix the beads in 50 μl of SureSelect Elution Buffer on a vortex mixer for 5 s to resuspend the beads.
13. Incubate the samples for 10 min at room temperature. Occasionally mix on a vortex mixer.
14. Briefly spin in a centrifuge, and separate the beads and buffer on a magnetic stand.
15. Transfer the supernatant (captured DNA) to a new 1.5-ml microfuge tube.
16. Add 50 μl of SureSelect Neutralization Buffer to the captured DNA and mix briefly.
17. Purify the sample using 180 μl of homogenous AMPure XP beads following the instruction and elute in 30 μl nuclease-free water.

Post-hybridization Amplification

1. Prepare the reaction mix on ice. Mix well on a vortex mixer.
2. Add 15 μl of DNA sample to 35 μl of the reaction mix.
3. Mix by pipetting, put the tube in a thermal cycler, and run the program
4. Purify the sample using 90 μl of homogenous AMPure XP beads and elute in 30 μl nuclease-free water following the instruction.
5. Assess quality and quantity.
6. Determine the concentration of the sample by integration under the peak.
7. The electropherogram should show a peak in the size range of approximately 300–400 nucleotides.
8. Do additional quantification by real-time PCR

Cluster Generation

1. Dilute and denature the libraries at 8 pM (∼1.6 pg/ul of 300 bp DNA) using 0.1 N NaOH.
2. Thaw and prepare the reagents following the instruction of Illumina Cluster Generation Kit.
3. Open and run appropriate recipe on the cluster station.
4. Follow recipe prompts to load flow cell.
5. Follow recipe prompts to load reagents.
6. Complete the cluster generation steps: hybridization, amplifi­cation, linearization, blocking, and primer hybridization.
7. Take the flow cell for sequencing.

Sequencing

1. Perform a pre-run wash step on sequencer.
2. Thaw and prepare sequencing reagent following the instruction from Sequencing Kit.
3. Load sequencing reagent.
4. Prime positions on the genome analyzer.
5. Clean and install prism and flow cell.
6. Check for proper reagent delivery and apply oil.
7. Perform read 1 first-base incorporation and auto-calibration.
8. Check quality metrics.
9. Continue the run for desired number of cycles.

Reference:

1. Priya R R, Rajasimha H K, Brooks M J, et al. Exome sequencing: capture and sequencing of all human coding regions for disease gene discovery. Retinal Development: Methods and Protocols, 2012: 335-351.