HLA-Typing by Next Generation Sequencing Protocol

Custom Oligo Library Amplification and T7 Promotor Addition

Custom Oligo Library Amplification (Optional)

1. Mix the following components in a sterile nuclease-free tube.

2. Run a PCR.

3. Vortex AMPure XP beads to resuspend.
4. Add 90 μL of resuspended beads to the PCR reactions. Mix by pipetting up and down at least 15 times.
5. Incubate for 5 min at room temperature.
6. Quickly spin the tube and place it on a magnetic stand to separate beads from the supernatant. Incubate at room temperature until the beads completely cleared from solution. Carefully remove and discard the supernatant. Do not discard the beads.
7. Add 200 μL of 70% ethanol to the PCR plate while in the magnetic stand. Incubate at room temperature for 1 min, and then carefully remove and discard the supernatant.
8. Repeat the last step once more.
9. Air dry the beads for 5 min while the PCR plate is on the magnetic stand with the lid open. Remove any residue liquid with
a pipette.
10. Remove the tube from the magnet. Elute DNA target from beads into 42 μL Elution Buffer (EB) or 0.1× TE. Pipet up and down at least 15 times. Quickly spin the tube and incubate at room temperature for 2–3 min.
11. Place the sample on an appropriate magnetic stand to separate beads from the supernatant. After the solution is clear, carefully transfer 40 μL supernatant to a new PCR tube. Samples can be stored at 2–8 °C for a few days or at −20 °C for long-term storage.
12. Check the size of product.

T7 Promotor Addition

1. Mix the components of Table 3 in a sterile nuclease-free tube.
2. Run a PCR.
3. To clean up and perform size selection of the PCR product vortex AMPure XP beads to resuspend.
4. Add 50 μL (1×) resuspended AMPure XP beads to 50 μL DNA solution. Mix well by vortexing or pipetting up and down at least 20 times.
5. Incubate for 5 min at room temperature.
6. Place the tube on a magnetic rack to separate the beads from the supernatant. After the solution is clear, carefully transfer the supernatant to a new tube and do not discard the supernatant.
7. Add 30 μL (0.6×) resuspended AMPure XP beads to the supernatant, mix well, and incubate for 5 min at room temperature.
8. Put the tube on a magnetic rack to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

9. Add 200 μL of 80% freshly prepared ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
10. Repeat step 9 once.
11. Keeping the tube on the magnetic rack, air dry the beads for 5 min.
12. Remove the tube from the magnet. Elute DNA target from beads into 32 μL Elution Buffer (EB) or 0.1× TE. Mix well on
a vortex mixer or by pipetting up and down, incubate for 2 min at room temperature.
13. Put the tube in a magnetic rack until the solution is clear, approximately 3 min. Transfer approximately 30 μL of the supernatant to a clean tube. Samples can be stored at 2–8 °C for a few days or at −20 °C for long-term storage.
14. Check the size of product.

RNA Bait Synthesis from DNA Template (Biotin-UTP Transcription)

1. Thaw the kit components, mix and pulse-spin in microfuge to collect solutions to bottom of tubes. Keep on ice.
2. Assemble the reaction
3. Incubate reaction at 37 °C for 14 h overnight.
4. To 20 μL reaction add 70 μL nuclease-free water, 10 μL of 10× Reaction Buffer, and 2 μL of RNase-free TURBO TM DNase, mix and incubate at 37 °C for 15 min.
5. Clean up RNA baits using Qiagen RNeasy MinElute Cleanup Kit according to the manufacturer's protocol. Split the reaction volume and use 50 μL per column. Elute twice in 15 μL and 10 μL of sterile nuclease-free water. Total elute volume is 25 μL.
6. Add 1 μL RNase inhibitor into eluted RNA baits.
7. Check the size distribution and concentration.
8. Adjust the concentration of baits to 100 ng/μL with very clean nuclease-free water (use separate tube of nuclease-free water only for this purpose). The concentration of 100 ng/μL is considered as stock solution of HLA RNA baits.

Quality Control of RNA Bait Library

Reverse Transcription (cDNA Synthesis)

1. Mix and briefly centrifuge each component of kit before use and combine the components in a 0.2 mL tube.

2. Incubate the tube at 65 °C for 5 min, then place on ice for at least 1 min.
3. Prepare the cDNA Synthesis Mix.

4. Add 10 μL of cDNA Synthesis Mix to RNA baits/primer mixture, mix gently and collect by brief centrifugation.
5. Collect the reaction by brief centrifugation. Add 1 μL of RNase H to each tube and incubate the tubes for 20 min at 37 °C. cDNA synthesis reaction can be stored at −20 °C for longterm storage or used for qPCR immediately.

SYBR Green qPCR

1. Mix the components in the sterile optical 96-well plate for real time PCR.

2. Run the reactions.

3. If synthesis of RNA baits was successful, CT values should appear between 10 and 15 cycles. NC should not show up at all or at >30 cycles.

Library Preparation

gDNA Fragmentation

1. To perform the NEBNext End Prep mix the components in a sterile nuclease-free tube.

2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
3. Place in a thermocycler, with the heated lid on, and run the program

4. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
5. Incubate at 20 °C for 15 min in a thermal cycler.
6. Add 3 μL Enzyme to the ligation mixture from last step.
7. Mix well and incubate at 37 °C for 15 min.
8. Clean-up of adaptor-ligated DNA.
9. Add 86.5 μL resuspended AMPure XP Beads to the ligation reaction. Mix well by pipetting up and down at least 10 times.
10. Incubate for 5 min at room temperature.
11. Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from the supernatant. After the solution is clear (about 5 min), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
12. Add 200 μL of 80% freshly prepared ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
13. Repeat step 12 once.
14. Air dry the beads for 5 min while the tube is on the magnetic stand with the lid open.
15. Remove the tube/plate from the magnet. Elute the DNA target from the beads by adding 17 μL of 10 mM Tris-HCl or 0.1× TE.
16. Mix well by pipetting up and down, or on a vortex mixer. Incubate for 2 min at room temperature.
17. Quickly spin the tube and place it on the magnetic stand.
18. After the solution is clear (about 5 min), transfer 15 μL to a new PCR tube for amplification.
19. Mix the components in sterile strip tubes to start PCR Amplification including indexing. Run the PCR program.
20. Add 45 μL of resuspended AMPure XP Beads to the PCR reactions (~50 μL). Mix well by pipetting up and down at least 10 times.
21. Incubate for 5 min at room temperature.
22. Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from the supernatant. After the solution is clear (about 5 min), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
23. Add 200 μL of 80% ethanol to the PCR plate while in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
24. Air dry the beads for 5 min while the PCR plate is on the magnetic stand with the lid open.
25. Remove the tube/plate from the magnet. Elute DNA target from beads into 33 μL 0.1× TE. Mix well by pipetting up and down at least 10 times. Quickly spin the tube and incubate at room temperature for 2 min.
26. Place the sample on an appropriate magnetic stand to separate beads from the supernatant. After the solution is clear (about 5 min), carefully transfer 28 μL supernatant to a new PCR tube. Libraries can be stored at −20 °C.
27. Check the size distribution with an aliquot of the library.

Targeted Enrichment (Bait-Based)

Hybridization

1. For singleplex hybridization (500 ng of DNA input), dilute HLA RNA baits stock solution (100 ng/μL) 1:5 with nuclease-free water.
2. For multiplex hybridization:
(a) 8 samples (62.5 ng of DNA input of each), dilute HLA RNA baits stock solution 1:5 with nuclease-free water.
(b) 6 samples (31.25 ng of DNA input of each), dilute HLA RNA baits stock solution 1:5 with nuclease-free water.
3. Set up the program on a thermocycler. This program will help during the pre-warming of the components and eventually perform the 65 °C incubation for hybridization.
4. Prepare the library mix in a nuclease-free tube and vortex.
5. Prepare the hybridization mix in a nuclease-free tube and vortex.
6. Prepare the capture mix in a nuclease-free tube and vortex.
7. Place the library mix in a thermocycler. Once cycle step 2 is reached, place the tube with the hybridization mix in the cycler to perform pre-warming. When step 3 of the cycler got reached, add the capture mix tube to the thermocycler to perform pre-warming of this component as well. Cycler step 4 represents the hybridization. When this step is reached add 7 μL of the pre-warmed library mix and 13 μL of the pre-warmed hybridization mix to the pre-warmed capture mix. Mix by gentle pipetting.
8. Incubate the hybridization reaction at 65 ° C for 36 h.

Recovery of Bait-DNA-Hybrid

1. Transfer 50 μL of magnetic beads to a new 1.5 mL tube.
2. Pellet beads using a magnetic particle stand and discard the supernatant.
3. Add 200 μL Binding Buffer to beads to wash. Vortex the tube for 5–10 s, place on a magnetic particle stand for 2 min to pellet the beads and remove and discard the supernatant.
4. Resuspend the beads in 200 μL Binding Buffer.
6. Transfer the hybridization solution to the Binding Buffer/ Beads and incubate for 30 min at room temperature. Pellet beads with magnetic particle stand for 2 min and remove the supernatant.
7. Add 500 μL Wash Buffer 1 to the beads and briefly vortex to resuspend. Incubate for 15 min at room temperature. Pellet beads with magnetic particle stand for 2 min and remove the supernatant.
8. Add 500 μL 65 °C Wash Buffer 2 to the beads and briefly vortex to mix. Incubate for 10 min at 65 °C. Pellet beads with magnetic particle stand for 2 min and remove the supernatant.
9. Repeat step 8 twice for a total of three 65 °C washes. Make sure all additional buffer is removed.

Elution of Target DNA

1. Add 50 μL freshly prepared Elution Buffer to beads.
2. Vortex for 5–10 s to mix.
3. Incubate for 10 min at room temperature.
4. Pellet the beads and transfer the supernatant to a tube containing 70 μL Neutralization Buffer.

AMPure XP Bead Purification

1. Add 120 μL AMPure XP Beads and incubate 5 min at room temperature.
2. Incubate for 5 min at room temperature.
3. Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
4. Add 200 μL of 80% ethanol while tube is in the magnetic stand. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
5. Dry 5 min at room temperature.
6. Remove the tube from the magnet. Add 30 μL 0.1× TE. Mix well by pipetting up and down at least 10 times. Quickly spin the tube and incubate at room temperature for 2 min.
7. Place the sample on an appropriate magnetic stand to separate beads from the supernatant. After the solution is clear, carefully transfer 28 μL of the supernatant to a new PCR tube.

Amplification of Enriched Target

1. Prepare PCR Mix on ice in a nuclease-free tube and mix by pipetting.
2. Place the tubes in a thermocycler and run the program.
3. Purify the DNA with AMPure XP Beads.
4. Validate and quantify an aliquot of the enriched library.

NGS Sequencing of Enriched Library

1. Load the HiSeq2500 with the enriched library and perform a paired-end sequencing using HiSeq® SBS Kit v4 (250 cycles).
2. Provide the demultiplexed fastq files from the sequencing run for the data analysis.

Reference:

1. Wittig M, Juzenas S, Vollstedt M, et al. High-resolution HLA-typing by next-generation sequencing of randomly fragmented target DNA[M]//HLA Typing. Humana Press, New York, NY, 2018: 63-88.