Full-Length HLA Genotyping with Nanopore Sequencing Protocol

PCR and Pre-Library Steps

1. Perform individual long-range PCRs with an appropriate long-range PCR kit with 50–200 ng genomic DNA and primers to amplify HLA class I genes. Verify by gel-electrophoresis.
2. Perform MID-PCRs with an appropriate long-range PCR kit using distinct barcodes/MIDs for each reaction.
3. Pool up to 96 MID-PCR products. Optionally, PCR product quantity may be equalized by adjusting the volume of pooled PCR product based on the gel-electrophoresis band intensities.
4. Purify 400 μl of the amplicon pool with SPRIselect beads in a bead/DNA volume ratio 0.6×. Make sure the beads are suspended completely. Mix by inversion and spin down only for a short time such that the beads remain dispersed. Incubate for 5–10 min. Separate beads on a magnetic rack. Discard the supernatant. Perform a wash step with 500 μl 70% ethanol leaving the tube at the magnetic rack. Discard the supernatant. Repeat the wash step. Spin down shortly and separate the ethanol at the bottom of the tube from beads using a magnetic rack, and remove it completely. Air-dry the pellet for 2 min. Elute with 150 μl water and spin down shortly. Incubate for 5 min. Separate beads using the magnetic rack.
5. Verify the DNA concentration.

Library Preparation

1. Prepare the DNA end-repair/dA-tailing reaction according to the manufacturer's guidelines.
2. Inactivation of the DNA end-repair/dA-tailing reaction by heat, and spin down shortly and transfer 100 μl to a 1.5 ml tube. Add 60 μl 0.6×of SPRIselect beads for purification to the DNA end-repair/dA-tailing reaction.
3. Quantify recovery by fluorescence. Ensure recovery of 700 ng to 1 μg of DNA.
4. Mix 22.5 μl of the eluted amplicon pool with 2.5 μl 1D adapters (ONT kit) and 25 μl Blunt/TA Ligase Master Mix and incubate for 10 min. Use water to reach a total volume of 100 μl and then add 60 μl (0.6×) SPRIselect beads.
5. Elute with 46 μl water. Incubate for 5 min. Use the magnetic rack for separation and transfer the supernatant to a fresh tube.
6. Add 5 μl BAM (ONT kit) and 50 μl Blunt/TA Ligase Master Mix, and incubate for 10 min.
7. Purify by adding 60 μl (0.6×) SPRIselect beads. Incubate for 5–10 min before separation. Discard the supernatant. Wash with 140 μl of "Adapter Bead Binding" buffer (ONT kit). Discard the supernatant. Repeat the wash step with 140 μl of "Adapter Bead Binding" buffer.
8. Air-dry the pellet for 2 min and then elute with 15 μl elution buffer (ONT kit). Incubate for 5 min and transfer the supernatant to a fresh tube after separation.
9. Quantify recovery by fluorescence. Ensure recovery of more than 250 ng of DNA. The library is now ready for sequencing and should be kept on ice.

Flow Cell Quality Control, Priming, and Sequencing

1. Place the flow cell in a MinION MK1B and connect the MinION to the computer on which the MinKNOW GUI is installed.
2. Label your experiment in the MinKNOW GUI, e.g., Sample ID and flow cell ID, and press the "Submit" button. Select Platform Quality Control (QC) script under "Choose operation" and press the "Execute" button. When the check is complete, the software will return to the start page. To see the active pore report, click on the "notification panel." The number of active pores should be more than 800.
3. Inspect the flow cell for air bubbles in the channel between priming port and array. To remove air bubbles, open the "Priming Port" and draw out buffer and bubbles; while removing buffer ensures that the sensor array is always covered by buffer.
4. Mix 480 μl Running Buffer (RBF) (ONT kit) with 520 μl water. Add 800 μl of the freshly prepared priming solution to the "Priming Port." Close the "Priming Port" and wait for 5 min. Open the "SpotON Sample Port" and add 200 μl to the "Priming Port". Do not introduce air bubbles while pipetting.
5. In a fresh tube mix 35 μl Running Buffer (RBF), 25.5 μl Loading Beads, 2.5 μl water, and 12 μl of the sequencing library. Dropwise, add 75 μl of this mix in the "SpotOn Sample Port." Replace the SpotON Sample Port cover and close the Priming Port. Close the MinION lid.
6. Select the appropriate protocol script under "Choose Operation" and launch sequencing by pressing the "Execute" button. The GUI will keep you updated on sequencing progression; the time required for sequencing being dependent on the chosen sequencing protocol.

FASTQ Extraction and Demultiplexing

1. Create a directory to store the FASTQ files.
2. Run albacore.
3. Download the demultiplexing scripts and copy the newly created FASTQ file into the same directory.
4. Make the wrapper script executable.
5. Run the demultiplexing routine.

Genotyping

1. Open NGSengine.
2. Open a "New project" via the file-button in the left-hand corner at the top.
3. Define a name for your project, and press "Next."
4. Add the folder for storing the demultiplexed fastq files, and press "Next."
5. A summary is shown, press "Next" to see a data overview for further analysis.
6. Open "Preferences" via the File tab in the left-hand corner at the top:
   (a) Within the "General" section, set the maximum number of reads to 10,000, set platform to "Oxford
   Nanopore" as the sequencing platform.
   (b) Within the "Locus default settings" section, set "Amplicon" to "Auto," and "Analysis region" to "Amplicon." Exclude
   problematic stretches, such as homopolymers, by excluding them in the "Ignore regions" field after setting it to "Custom." Use the IPD-IMGT/HLA genomic coordinate system to set the regions.
   (c) Within the "Locus Selection" section, define the loci that were in the sequencing run. Put them in the "Analysis" column.
7. Define the Loci by holding the mouse over the sample to be analyzed; via right mouse click, set the HLA locus/loci that should be typed.
8. Press "Analyze" for all samples at the left-hand site of the screen or for a specific sample at the right-hand side at the end of the sample line.
9. When the data analysis is complete, an overview of the genotyped alleles for the samples is shown.

Reference:

1. Lang K, Surendranath V, Quenzel P, et al. Full-length HLA class I genotyping with the MinION nanopore sequencer[M]//HLA Typing. Humana Press, New York, NY, 2018: 155-162.