Full-Length HLA Typing Method Using PacBio SMRT Sequencing Protocol

HLA Amplicon Generation

DNA Extraction

1. Pipet 20 μl proteinase K into the bottom of a 1.5 ml microcentrifuge tube.
2. Add 200 μl blood sample followed by 200 μl Buffer AL to the microcentrifuge tube. Mix the sample by pulse-vortexing for 15 s.
3. Incubate at 56 °C for 10 min.
4. Spin down the contents and add 200 μl ethanol (96-100%) to the sample.
5. Mix again by 15 s pulse-vortexing followed by spinning down to remove drops from the inside of the lid.
6. Centrifuge at 6000 × g (8000 rpm) for 1 min.
7. Discard the collection tube containing the filtrate and place the spin column in a clean 2 ml collection tube.
8. Add 500 μl Buffer AW1 without wetting the rim and centrifuge at 6000 × g (8000 rpm) for 1 min.
9. Place the spin column in another clean 2 ml collection tube, and discard the collection tube containing the filtrate.
10. Add 500 μl Buffer AW2 to the column without wetting the rim. Close the cap and centrifuge at full speed (20,000 × g; 14,000 rpm) for 3 min.
11. Again place the spin column in a new 2 ml collection tube and discard the old collection tube with the filtrate.
12. Centrifuge the column at full speed for 1 min.
13. Place the spin column in a clean 1.5 ml microcentrifuge tube, and discard the collection tube containing the filtrate.
14. Add 200 μl Buffer AE or distilled water. Incubate at room temperature (15-25 °C) for 1 min, and then centrifuge at 6000 × g (8000 rpm) for 1 min.

Assessment of DNA Quality and Quantity

1. Analyze the quality of DNA on 1% w/v agarose gel using 3 μl of extracted DNA.
2. Quantify the DNA using 1 μl of DNA and analyze as per manufacturer's protocol.

PCR Amplification of Full-Length HLA Genes

1. All the reactions must be set on ice.
2. Thaw 10× Long Range PCR Buffer, dNTP mix, nuclease-free H₂O, and primer solutions. Mix the solutions thoroughly and centrifuge briefly before use.
3. Prepare a reaction mix. Prepare a separate reaction mix for each amplification primer.
4. HLA-DQB1 requires the addition of Q-Solution and double the amount of Long-Range enzyme per reaction.
5. Vortex the reaction mix thoroughly, and centrifuge briefly.
6. Add the reaction mix into each PCR tube. The appropriate volume is 25 μl minus the amount of DNA added in the next step.
7. Add 1-4 μl template DNA (50-200 ng) to each tube containing reaction mix.
8. Program the thermal cycler according to the manufacturer's instructions.
9. After amplification, store the samples overnight at 2-8 °C

Quality Assessment of PCR Amplicon

1. Quantify the amplicons of class I and II genes using 1 μl of PCR assay and analyze as per manufacturer's protocol.
2. Analyze the PCR products on 1% w/v agarose gel using 3 μl of each PCR assay.
3. Confirm the size of amplicon by PCR.

PCR Product Purification

1. All amplicons of class I and II genes were purified using kit.
2. Keep the AMPure beads at room temperature for half an hour.
3. Add 0.6× of AMPure beads (e.g., for 100 μl add 60 μl of AMPure beads) to the PCR amplicon and mix by pipetting up and down.
4. Spin down the tube to collect the beads and keep it at room temperature for 15 min.
5. Keep the tube in a magnetic bead rack until the beads collect to the side of the tube and the solution appears clear.
6. Keep the tubes on the magnetic bead rack, and remove the cleared supernatant to another tube by pipetting off.
7. Do not remove the tube from the magnetic bead rack and add freshly prepared 70% ethanol to the Eppendorf tube to the opposite side of beads.
8. Use a sufficient volume of 70% ethanol to fill the tube without disturbing the beads. Let the tube sit for 30 s.
9. After 30 s, pipette and discard the 70% ethanol.
10. Repeat steps 6-8 above.
11. Remove residual 70% ethanol by short spin and place back on magnetic stand.
12. Pipette off any remaining 70% ethanol.
13. Allow beads to air-dry (with the tube caps open) for 30-60 s.
14. Remove the tube from the magnetic rack and add 50 μl of elution Buffer to the sample. Elute the DNA off the beads.
15. Vortex for 30 s and incubate at room temperature for 10 min.
16. Put the tube back on the magnetic rack to elute the sample from the beads and transfer the sample to a fresh tube.

Quality Assessment of Purified PCR Amplicon

1. Quantify the purified amplicons of class I and II genes using 1 μl of DNA and analyze as per manufacturer's protocol.
2. Check the size of amplicon and reagents as per the manufacturer's protocol.
3. Confirm the size of amplicons as per the expected size.

HLA SMRTbell Library Preparation

1. Prepare the SMRTbell library using SMRT template preparation kit as per the manufacturer's protocol. SMRT template preparation protocol consists of the following steps.
2. Equimolar pooling of purified PCR amplicon.
3. Repair DNA damage.
4. Purify the repaired DNA.
5. Blunt-end ligation of SMRTbell adapters to the end-repaired amplicons.
6. Add exonuclease and incubate.
7. Purify SMRTbell templates.
8. SMRTbell Library Quality Assessment.
9. Anneal primers to SMRTbell templates.
10. Bind polymerase to SMRTbell templates.

Equimolar Pooling of Purified PCR Amplicon

1. Pool equal molar concentration of HLA-A, HLA-B, HLA-C, HLA-DQB1 and HLA-DRB1 as one sample.
2. Keep target volume of final pool (μl) as 20 μl and target concentration per amplicon (nM) for equimolar pool as 4 nM.

Repair DNA Damage in Amplicons

1. Thaw the kit component on ice and add the reagents in a microfuge tube.
2. Mix the content by pipetting and spin down tube contents.
3. Incubate at 37 °C for 20 min or longer, then return reaction to 4 °C for 1 min.

Purify the Repaired DNA

1. Bring the bead reagent to room temperature and mix it well until the solution appears homogenous.
2. Add 0.6× volume of AMPure PB magnetic beads to the End-Repair reaction and mix by pipetting up and down.
3. Spin (or pulse) down the tube to collect the beads and transfer the tube to a vortex mixer.
4. Allow the DNA to bind to beads by shaking at 2000 rpm for 10 min at room temperature until the bead/DNA mixture should appear homogenous.
5. Spin down the tube for a few seconds to collect beads.
6. Place the tube in a magnetic bead rack until the beads collect to the side of the tube and the solution appears clear.
7. Keeping the tubes on the magnetic bead rack, remove the cleared supernatant to another tube by pipetting off.
8. Do not remove the tube from the magnetic bead rack and add freshly prepared 70% ethanol to the Eppendorf tube to the opposite side of beads.
9. Use a sufficient volume of 70% ethanol to fill the tube without disturbing the beads. Let the tube sit for 30 s.
10. After 30 s, pipette and discard the 70% ethanol.
11. Repeat steps 6-8 above.
12. Remove residual 70% ethanol by short spin and place back on magnetic stand.
13. Pipette off any remaining 70% ethanol.
14. Allow beads to air-dry (with the tube caps open) for 30-60 s.
15. Remove the tube from the magnetic rack and add 34 μl of Elution Buffer to the sample. Elute the DNA off the beads.
16. Vortex for 10 min at 2000 rpm.
17. Put the tube back on the magnetic rack to elute the sample from the beads and transfer the sample to a fresh tube.
18. The End-Repaired DNA can be stored overnight at 4 °C or at −20 °C for longer duration.

Blunt-End Ligation of SMRTbell Adapters to the End-Repaired Amplicons

1. Thaw the reagents on ice and mix them in a microcentrifuge tube as the manufacturer's protocol.
2. Add the adapter to the DNA whereas all other components should be added to the Master Mix. Make up the total volume to 40 μl with water.
3. Mix the reaction well by pipetting and spin down the tube contents.
4. Incubate at 25 °C for 15 min.
5. Incubate at 65 °C for 10 min to inactivate the ligase and store at 4 °C.
6. Don't stop and proceed with adding exonuclease after this step.

Add Exonuclease and Incubate

1. Thaw the reagent on ice and mix the reaction well by pipetting.
2. Spin down the tube contents with a quick spin in a microfuge.
3. Add the reagents as the manufacturer's protocol, mix by pipetting and spin down.
4. Incubate at 37 °C for 1 h, then return the reaction to 4 °C.

SMRTbell Library Quality Assessment

1. Quantify the libraries using 1 μl of DNA as per the manufacturer's protocol.
2. Check the size of library.

Anneal Primers to SMRTbell Templates

1. In the calculator, click on number of SMRT Cells option which specifies how many SMRT Cells to prepare, and the Calculator determines the amount of sample necessary.
2. Add the library concentration and size of libraries.
3. Protocol: Select the loading method as MagBead.
4. Binding Kit: Select the sequencing polymerase as P6v2.
5. Preparation Protocol: Small scale.
6. Long Term Storage: No.
7. DNA Control Complex: Yes.
8. Complex Reuse: No.
9. Standard Concentration: Yes
10. Click on Custom Parameters section.
11. Concentration on Plate: Use the default recommendation as 0.01 nM.
12. DNA Control Complex Ratio to Template: Use the default recommendation as 1.2%.
13. Polymerase: Template Ratio: Use the default recommendation as 10:1.
14. Primer: Template Ratio: Use the default recommendation as 20:1.
For the Conditioning primer:
(a) Dilute and preheat the Sequencing Primer from 5000 to 150 nM in Elution Buffer.
(b) Incubate the diluted primers at 80 °C for 2 min then hold at 4 °C.
For the annealing primer:
(a) In a fresh tube, add the appropriate amount of reagents.
(b) Mix the content with pipetting, spin down briefly, and incubate at 20 °C for 30 min.
(c) Transfer to 4 °C location for immediate use or store at −20 °C.

Bind Polymerase to SMRTbell Templates

1. Thaw the reagents for polymerase dilution and prepare the reaction on ice.
2. Dilute the polymerase in 0.5 ml tube, mix by pipetting, and spin down briefly.
3. For polymerase binding, add the components, mix by pipetting and spin down briefly.
4. Incubate at 30 °C for 30 min and hold at 4 °C.

Bind the DNA Complex to MagBead

1. Dilute the DNA Internal Control Complex.
2. Further dilute the first dilution of DNA control.
3. Dilute the sample complex.
4. Keep the magnetic beads cold.
5. Add 73.9 μl MagBeads to empty tube and place it on magnetic stand.
6. Collect beads. Remove the supernatant and discard.
7. Add 73.9 μl of MagBead wash buffer and wash by slowly aspirating and dispensing ten times.
8. Collect beads. Remove the supernatant and discard.
9. Add 73.9 μl of MagBead Binding Buffer and mix by slowly aspirating and dispensing ten times.
10. Add 73.9 μl of washed beads to a new tube and place it on a magnetic stand.
11. Collect beads. Remove the supernatant and discard.
12. Add 19 μl of diluted sample complex and mix by slowly aspirating and dispensing ten times.
13. Incubate in a rotator at 4 °C for 20 min (up to 2 h).
14. Keep the tube containing MagBead complex on a magnetic plate.
15. Collect beads. Remove clear supernatant and discard.
16. Add 19 μl of MagBead binding buffer and mix by slowly aspirating and dispensing ten times.
17. Collect beads by placing the tube on magnetic stand. Remove the supernatant and discard.
18. Add 19 μl of MagBead wash buffer and wash by slowly aspirating and dispensing ten times.
19. Collect beads by placing the tube on magnetic stand. Remove the supernatant and discard.
20. Add 1.2 μl of DNA Control Dilution.
21. Add 17.8 μl of MagBead Binding Buffer and mix by slowly aspirating and dispensing ten times.
22. Keep at 4 °C until use.

Sequencing

1. Loading the sample plate: Transfer the specified volumes (19 μl) into separate well of a new 96-well PCR plate for processing on PacBio RS.
2. Load the reagents, tips and SMRT cells on the sequencer and start sequencing: The reagents and SMRT cell vary with type of sequencer used, therefore refer to for details.

Reference:

1. Ambardar S, Gowda M. High-resolution full-length HLA typing method using third generation (Pac-Bio SMRT) sequencing technology[M]//HLA Typing. Humana Press, New York, NY, 2018: 135-153.