1. Total RNA isolation from whole blood or cells
[Maxwell 16 LEV simplyRNA Purification Kit (Promega); alternatively other RNA isolation protocols may be utilized - other methods that we have tested: RNA-DNA isolation out of TRIzol; RNA isolation from PAXgene blood tubes]
(a) Prepare the whole blood/PBMC samples:
(1) Rotate fresh (not frozen) whole blood sample in EDTA tube on a mixer.
(2) Transfer 2.5 ml mixed whole blood to a 15 ml tube.
(3) Add 7.5 ml Cell Lysis Solution, and invert the tube 5-6 times to mix.
(4) Incubate for 10 min at RT. Twice during incubation, invert to mix.
(5) Centrifuge for 10 min at 3000 x g. Remove and discard the supernatant without disturbing the pellet. Briefly spin to collect any liquid from the sides of the tube to the bottom, and again remove and discard the supernatant.
(6) Add 200 µl chilled 1-Thioglycerol/Homogenization Solution to the pellet (20 µl of 1-Thioglycerol per 1 ml Homogenization Solution). Pipet or vortex to mix to completely resuspend the pellet.
(7) Add 200 µl Lysis Buffer and 25 µl Proteinase K, and vortex for 20 s to resuspend the pellet.
(8) Incubate for 10 min at RT. Prepare the simplyRNA Blood Cartridges at this time.
(1) Thaw cells at room temperature on a mixer.
(2) The kit can process up to 5 million cells, so remove an appropriate aliquot for more concentrated samples.
(3) Centrifuge for 2 min at 3000 x g to pellet the cells. Remove the supernatant.
(4) Add 200 µl chilled 1-Thioglycerol/Homogenization Solution to the pellet (20 µl of 1-Thioglycerol per 1 ml Homogenization Solution).
(5) Pipet or vortex to mix to completely resuspend the pellet.
(6) Add 200 µl Lysis Buffer to lysed cells, vortex for 15 s to resuspend the pellet.
(7) Prepare the simplyRNA Cells Cartridges at this time.
(b) Prepare the Maxwell cartridges:
(1) Place the cartridges for the appropriate kit into the Maxwell 16 LEV Cartridge Rack with the label side facing away from the elution tubes. Press down on each cartridge to snap into place. Note: If processing less than 16 samples, center the cartridges in the rack.
(2) Carefully peel back the seal so that all plastic comes off the top of each cartridge.
(3) Place an LEV Plunger in well #8 of each cartridge (well closest to the elution tube).
(4) Place 0.5 ml Elution Tubes in the Cartridge Rack. Add 50 µl Nuclease-Free Water to the bottom of each elution tube. Note: This can be reduced down to as low as 30 µl water for a more concentrated eluate.
(5) Add 10 µl DNase I solution to well #4 of the each cartridge.
(6) Add lysate prepared above to well #1 of each cartridge.
(c) Follow the instrument’s on-screen prompts to select and run the appropriate protocol.
(d) Immediately upon completion of the run, remove isolated RNA elution tubes from the Maxwell and transfer samples to properly labeled storage tubes.
(e) Quantitate RNA samples with a NanoDrop (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80°C.
2. Synthesis of first-strand cDNA
[MJ Research Tetrad Thermocycler (Bio-Rad Laboratories), Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen); alternatively other thermocyclers and cDNA synthesis protocols may be utilized - other methods that we have tested: RevertAid First Strand cDNA Synthesis]
(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.
(b) Incubate reactions at 65°C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUT, and 1 µl SuperScript III RT.
(c) Incubate initial reactions at 4°C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.
(d) Incubate at 50°C for 50 min.
(e) Terminate cDNA synthesis by incubating at 85°C for 5 min.
(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37°C for 20 min to remove any remaining RNA.
(g) Upon completion, proceed directly to PCR or store cDNA at -20°C.
3. PCR amplification of full-length MHC class II cDNA amplicons
[Phusion High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs), FlashGel DNA Cassette 1.2% (Lonza), FlashGel 5X loading dye (Lonza), FlashGel Quant Ladder 100–1.5 kb (Lonza)]
(a) For multiplexed PacBio sequencing, each sample must be uniquely tagged with molecular barcodes to extract the reads per sample out of the pool of sequence reads. PacBio sequencing supports either symmetrical (forward barcode #1 with reverse barcode #1, etc.) or asymmetrical (forward barcode #1 with reverse barcode #2, etc.) barcoding protocols. Additional information about PacBio barcoding can be found on the Pacific Biosciences GitHub site. We routinely use fusion primers containing our MHC class I sequence-specific PCR primers tagged with PacBio barcodes 0001-0016. The sequences as ordered from IDT are in the PacBio MHC-II primers Excel file. For each different barcode, prepare separate 1 μM working stocks of the following 4 primer cocktails (for IDT plate orders normalized to 150 μM, use 1.5 μl of each primer plus nuclease-free water up to 150 μl total volume):
DRB-F DQB-F DPB-F
DRB-R DQB-R DPB-R
DRA-F DQA-F DPA-F
DRA-R DQA-R DPA-R
(b) Perform 6x PCR reactions per sample, one reaction per locus, to ensure a high enough final product concentration for PacBio sequencing (target: 30-100 ng/µl DNA in 30-200 µl volume for each final pool; 1-3 µg total DNA for 1-2 Kb library). Prepare 50 µl primer-specific reaction mixtures of 25 µl 2X Phusion High-Fidelity PCR Master Mix, 14 µl nuclease-free water, 5 µl 1 µM 'F' PCR primer, 5 µl 1 µM 'R' PCR primer, and 1 µl cDNA.
(c) PCR amplify using the following cycle conditions: 98°C for 3 min, 25 cycles of 98°C for 5 s, 60°C for 1 s, 72°C for 20 s, and a final elongation of 72°C for 5 min.
(d) After 25 cycles, check a 4 µl aliquot of each reaction on a FlashGel DNA cassette following the manufacturer’s protocol. Add additional cycles to reactions as needed to obtain sufficient amplification of each sample (typically 3 additional cycles is sufficient; should not exceed 30 cycles).
4. PCR product purification
[AMPure XP PCR Purification Kit (Beckman-Coulter)]
(a) Transfer 45 µl of each reaction to a 300 µl round bottom plate.
(b) Bring the AMPure XP beads up to RT. Gently shake the bottle to resuspend any magnetic particles that may have settled.
(c) Add 45 μl AMPure XP beads (1:1 reaction volume to bead volume). Pipet 10 times to mix.
(d) Incubate for 5 min at RT.
(e) Place reaction tube onto magnet plate for 2 min to pellet beads.
(f) Aspirate clear supernatant from the plate while still on magnet and discard.
(g) Add 200 μl of 70% ethanol to plate while still on magnet, incubate for 30 s at RT, then aspirate out ethanol and discard.
(h) Repeat step (g) for a second ethanol wash.
(i) Dry the pellets on the magnet for 5 min at RT.
(j) Remove the plate from the magnet. Add 40 μl of TE buffer and pipet 10 times to resuspend the beads.
(k) Incubate for 2 min at RT.
(l) Place the plate back on the magnet for 1 min to pellet the beads, and transfer supernatant to a new plate (make sure no beads are transferred).
(m) Repeat steps (c)-(l) for a second AMPure XP clean-up, using 40 μl AMPure XP beads in step (c) and resuspending in 30 μl of TE buffer in step (l).
5. Quantitation of amplicon products
[Quant-iT dsDNA HS Assay Kit (Invitrogen), Qubit assay tubes (Invitrogen); alternatively other fluorescence-based methods may be utilized, especially when dealing with large numbers of samples (use of a Nanodrop or other spectrophotometer is not recommended at this stage) - other methods that we have tested: Plate reader quantification with PicoGreen]
(a) Bring all reagents to RT.
(b) Prepare the Quant-iT working solution of 199 µl Quant-iT buffer and 1 µl Quant-iT reagent.
(c) Dilute the standards by adding 10 µl standard to 190 µl Quant-iT mix (1:20 dilution).
(d) Dilute the samples by adding 2 µl sample to 198 µl Quant-iT mix (1:100 dilution).
(e) Vortex each sample for 2-3 s. Incubate at RT for 2 min.
(f) Calibrate the fluorometer and read samples as directed by the manufacturer's protocol.
(g) Calculate concentrations of samples: concentration = QF x (200/sample volume) [conc = QF/10 when using 2 µl sample].
6. Pooling, purification, and quantitation of the PCR amplicon pool
[AMPure XP PCR Purification Kit (Beckman-Coulter), Quant-iT dsDNA HS Assay Kit (Invitrogen), Qubit assay tubes (Invitrogen)]
(a) Calculate the volume required to obtain 250 ng of each DRB sample and 80 ng of each sample for the other 5 loci (DRA, DQB, DQA, DPB, DPA) – since there are 4-6 DRB alleles expected per sample, but only up to 2 alleles per sample at each of the other 5 loci, the DRB amplicons should be more concentrated in the pool. Target concentrations can be reduced if necessary, but the pool should contain >3 μg total DNA, as there will be a reduction in final concentration after additional AMPure XP purification of the pool.
(b) Add the calculated volume of each sample to a 1.5 ml tube to create a pool of uniquely barcoded PCR products. Determine the total volume of the pool.
(c) Bring the AMPure XP beads up to RT. Gently shake the bottle to resuspend any magnetic particles that may have settled.
(d) Add an equal volume of AMPure XP beads to the pool of PCR products. Pipet 10 times to mix.
(e) Incubate for 5 min at RT.
(f) Place reaction tube onto magnet stand for 2 min to pellet beads.
(g) Aspirate clear supernatant from the tube while still on magnet and discard.
(h) Add 1.5 ml of 70% ethanol to tube while still on magnet, incubate for 30 s at RT, then aspirate out ethanol and discard.
(i) Repeat step (h) for a second ethanol wash.
(j) Dry the pellet on the magnet for 5-10 min at RT.
(k) Remove tube from the magnet. Add 100 μl of TE buffer and pipet 10 times to resuspend the beads.
(l) Incubate for 2 min at RT.
(m) Place the tube back on the magnet for 1 min to pellet the beads, and transfer supernatant to a new tube (make sure no beads are transferred).
(n) Repeat steps (d)-(m) for a second AMPure XP clean-up, using 100 μl AMPure XP beads in step (d) and resuspending in 50 μl of TE buffer in step (k).
(o) Read duplicate 2 μl aliquots of the final pool on the Qubit following the directions in 5. Quantitation of amplicon products.
(p) Pools of products are now ready for submission to a PacBio sequencing facility for library preparation and sequencing.
7. Sequence analysis
A semi-automated pipeline for PacBio sequence analysis is under development.
An outline of the data analysis workflow is shown below. Conveniently, the same general workflow is applicable to MHC class I and MHC class II sequences with minimal modification:
Briefly, we first remove sequences containing insertion/deletion base calling artifacts (indels). The predominant type of sequencing error in PacBio reads are randomly distributed indels. In our preliminary experiments with P6/C4 chemistry, approximately 80% of reads have one or more indels. Therefore, we conceptually translate each sequencing read in all six reading frames and retain only those sequences containing an open reading frame of the expected size for each locus.
Next, we compare each read to a library of known alleles to detect chimeric sequences (using the UCHIME algorithm; see http://drive5.com/usearch/manual/uchime_algo.html). Depending on the sample, 10-50% of reads may be discarded during these aggressive filtering steps to remove PCR artifacts.
Non-chimeric sequences are then mapped to a full length allele reference database. Those reads mapping to know allele sequences at 100% identity are saved. Those reads not mapping continue on in the analysis pipeline.
Identical sequences are clustered; clusters supported by at least 3 independent reads are retained. The threshold of three identical PacBio reads meets newly updated standards for allele assignment from deep sequencing established by the IPD. Nucleotide mis-incorporations are most likely to occur in the last few cycles of a PCR reaction, so reads that are unique (that is, those that appear only once in a readset) are frequently PCR artifacts. We remove such unique reads from subsequent analyses by applying the cluster size filter of 3.
Representative cluster sequences are then mapped at 100% identity in the overlap to a reference allele database of partial length MHC sequences. Those sequences that map to partial MHC allele sequences are saved as sequence extensions and used to update existing database sequences to full length.
Presumptive, authentic transcripts not mapping to known sequences are annotated and curated.