Holotype HLA 24 7 RUO Quick Protocol Guide v2
PRE-PCR Holotype HLA 24/7 Quick Protocol Guide
Step 0 – Genomic DNA Preparation
o
Dilute all gDNAs to a concentration of 20 - 30ng/µL (minimum volume is 45 µL).
o
Thaw HLA-A, B, C, DRB1, DPB1, DQA1, DQB1 (set 1) and DQB1 (set 2) primer sets, Enhancer 1, Enhancer 2, Long-Range PCR Buffer (10X) and dNTP mix (10mM each) to room temperature.
o
Prepare the Combined Enhancer. Add 132 μL of Enhancer 2 directly into the Enhancer 1 tube, and relabel the tube as Combined Enhancer.
Step 1 – HLA Amplification Master Mix Preparation
o
Prepare a Master Mix for each HLA locus according to the following tables:
Master Mix Table for HLA-A, B, C, DRB1, DPB1 and DQA1 Reagent Primer Mix LongRange PCR Buffer (10×) dNTP Mix (10 mM each) Molecular grade H2O Total Volume
Volume/sample/locus 2 μL 2.5 μL 1.25 μL 13.85 μL 19.6 μL
Volume/24 samples/locus 51 μL 63.8 μL 31.9 μL 353.2 μL 499.9 μL
Master Mix Table for HLA-DQB1 Set 1 (Optional) and HLA-DQB1 Set 2 (Required) Reagent Primer Mix LongRange PCR Buffer (10×) dNTP Mix (10 mM each) Combined Enhancer Molecular grade H2O Total Volume
Volume/sample/locus 2 μL 2.5 μL 1.25 μL 5.6 μL 7.85 μL 19.2 μL
Volume/24 samples/locus 51 μL 63.8 μL 31.9 μL 142.8 μL 200.2 μL 489.7 μL
o
Vortex each Master Mix and spin down for 1 second. Place Master Mixes on ice.
Step 2 – HLA Class I and II Amplification
o
Remove the Taq Polymerase from storage, spin it down, and add it to each Master Mix according to the tables below, rinsing the pipette tips thoroughly by pipetting:
Taq Polymerase-Loaded Master Mix: HLA-A, B, C, DRB1, DPB1, and DQA1 Reagent Master Mix from Step 1 Taq Polymerase Total Volume
Volume/sample/locus 19.6 μL 0.4 μL 20 μL
Volume/24 samples/locus 499.9 μL 10.2 μL 510.1 μL
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
1
PRE-PCR Taq Polymerase-Loaded Master Mix: HLA-DQB1 Set 1 (Optional) and HLA-DQB1 Set 2 (Required) Reagent Master Mix from Step 1 Taq Polymerase Total Volume
Volume/sample/locus 19.2 μL 0.8 μL 20 μL
Volume/24 samples/locus 489.7 μL 20.4 μL 510.1 μL
o o
Briefly vortex and spin down all Taq Polymerase-loaded Master Mixes.
o
Add 5 μL of each diluted sample of gDNA into the designated wells of each locus-specific 96-well Amplification Plate.
o
Seal the plates with a thermal seal, centrifuge for 10 seconds, place the Class I and Class II Amplification Plates into separate thermal cyclers and run the following programs:
Aliquot 20 μL of the appropriate Taq Polymerase-loaded Master Mix for each locus into the Class I and Class II Amplification Plates.
Class I Amplification Number of Cycles 1 35 1 1
Class II Amplification
Temperature
Time
95°C 95°C 65°C 68°C 68°C 4°C
3 minutes 15 seconds 30 seconds 5 minutes 10 minutes ∞
Number of Cycles 1 35 1 1
Temperature
Time
95°C 93°C 60°C 68°C 68°C 4°C
3 minutes 15 seconds 30 seconds 9 minutes 10 minutes ∞
Safe stopping point. Amplicons can be stored at 4°C overnight or -20°C for longer periods.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
2
POST-PCR Step 3 – Amplicon Quantitation and Normalization Quick tip – A concentration of 50 ng/μL or greater is considered a successful amplification.
o o
Remove the QuantiFluor dsDNA dye system from the fridge and let it come to room temperature. Follow the dilution table below to create a serial dilution of the QuantiFluor Lambda DNA standard (100 ng/μL):
Label on tube Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Blank
Input DNA
Volume DNA (μL)
Lambda DNA Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Blank
7.5 μL 250 μL 250 μL 250 μL 250 μL 250 μL 0 μL
Volume 1x TE (μL) 492.5 μL 250 μL 250 μL 250 μL 250 μL 250 μL 250 μL
Final Conc. (ng/μL) 1.5 ng/μL 0.75 ng/μL 0.38 ng/μL 0.19 ng/μL 0.09 ng/μL 0.05 ng/μL 0 ng/μL
o
Dilute 1 μL of amplicon DNA from the Amplicon Plate into 99 μL 1× TE buffer in a 96-well optical plate. Seal the plate, vortex well and centrifuge plate for 10 seconds.
o
Prepare 1× QuantiFluor Dye working solution by mixing 0.5 μL QuantiFluor Dye (200X) + 99.5 μL 1× TE buffer per sample. Prepare sufficient 1× QuantiFluor Dye working solution so that each sample (total number of samples in Amplicon Plates) and standard (14 total) will receive a 100 µL aliquot.
o
Aliquot 100 μL of 1× QuantiFluor Dye working solution into the appropriate wells of two 96-well optical plates to prepare a Standards Quantitation Plate and an Amplicon Quantitation plate.
o
Add 100 μL of each standard, in duplicate, to individual wells in the Standards Quantitation Plate (14 wells total). Mix well by pipetting without creating bubbles. NOTE: If using a qPCR machine use qPCR machine-compatible plates and divide all volumes in half (combine 50 µL of Quantifluor working solution with 50 µL of diluted amplicons or 50 µL of standards).
o
Run the Standards Quantitation Plate on the plate fluorometer (or qPCR machine) followed by the Amplicon Quantitation Plate and calculate the concentration of DNA in the Amplicon Quantitation Plate using RFU data generated by the plate fluorometer (or qPCR machine).
o
Dilute DNA in the Amplicon Plate with molecular grade H2O so that the final concentration of DNA is approximately 67 ng/μL. • If DNA concentration is 150 ng/μL or greater: add 25 μL of H2O • If DNA concentration is 100-150 ng/μL: add 10 μL of H2O • If DNA concentration is less than 100 ng/μL: add 0 μL of H2O
o o
For each sample, combine 15 μL of the DQB1 (Set 1) and DQB1 (Set 2) into an unused well (only for SUCCESSFUL amplifications if both of DQB1 sets were used). Pool all loci for each sample into a single Amplicon Plate by combining 5 μL from each locus of a sample into a new 96-well PCR plate. Each sample should occupy a single well, with a volume of 35 μL.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
3
POST-PCR o o
Mix well by pipetting.
Aliquot 4 μL of ExoSAP-IT Express (Affymetrix) into each amplicon in the Class I and Class II Amplification Plates using a multichannel pipette and mix by pipetting. Seal the Amplification Plates with a thermal seal, centrifuge for 10 seconds, place them into a thermal cycler and run the following program: ExoSAP-IT Express Program Number of Cycles 1 1 1
Temperature 37°C 80°C 4°C
Time 4 minutes 1 minutes ∞
Safe stopping point. Amplicons can be stored at 4°C overnight or -20°C for longer periods.
Step 4 – Library Preparation
The reagents used during library preparation are very viscous and particular attention is required while pipetting them. *IMPORTANT* - Use PCR Freezer Blocks to setup the Fragmentation reactions. It’s very important to keep all the reagents cold at all times and to pre-heat the thermal cycler prior to putting the plate on it.
o o o
Thaw the Fragmentation Buffer to room temperature and keep it on ice. Vortex the Fragmentation enzyme thoroughly. Prepare the Fragmentation Master Mix on ice according to the following table and vortex the Fragmentation Mix to mix:
Reagent Fragmentation Enzyme (A) Fragmentation Buffer (B) Total volume
Volume per library (μL) 2 μL 2 μL 4 μL
Recommended volumes per 24 libraries (μL) 55.2 μL 55.2 μL 110.4 μL
Color code Yellow Red
o
Place a 96-well Reagent Plate on a PCR cooler block, aliquot the Fragmentation Master Mix in a clean column and spin for 10 seconds.
o
Place a 96-well Reaction Plate on a second PCR cooler block and aliquot 4 μL of Fragmentation Master Mix from the Reagent Plate into each well of the Reaction Plate that will be used with a multichannel pipette.
o
Replace the Reagent Plate with the Amplicons Plate on the PCR cooler block and transfer 16 μL of each amplicon from the Amplicons Plate to the corresponding well on the Reaction Plate using a multichannel pipette. Mix by pipetting.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
4
POST-PCR o
Cover the Reaction Plate with a thermal seal, centrifuge for 10 seconds and incubate the Reaction Plate in a thermal cycler with the following program: Fragmentation Program Number of Cycles 1 1 1
Time 10 minutes 15 minutes ∞
Safe stopping point. Products can be stored at 4°C overnight or -20°C for longer periods.
o o
Temperature 37° C 70° C 4° C
Thaw the End Repair Buffer to room temperature and keep it on ice. Prepare the End Repair Master Mix on ice according to the following table and mix by pipetting:
Reagent
Volume per library (μL)
Molecular grade H2O End Repair Enzyme (C) End Repair Buffer (D) Total volume
1.25 μL 1.25 μL 2.5 μL 5 μL
Recommended volumes for 24 libraries (μL) 34.8 μL 34.8 μL 69.6 μL 139.2 μL
Color code Green Orange
o
Aliquot the End Repair Master Mix in a clean column of the Reagent Plate and transfer 5 μL into each well of the Reaction Plate using a multichannel pipette. Mix thoroughly by pipetting.
o
Cover the Reaction Plate with a thermal seal, centrifuge for 10 seconds and incubate the Reaction Plate in a thermal cycler with the following program:
End Repair Program Number of Cycles 1 1 1
Temperature 20°C 65°C 4°C
Time 30 minutes 30 minutes ∞
Safe stopping point. Products can be stored at 4°C overnight or -20°C for longer periods.
o
Remove Indexed Adaptor Plate from -20°C and allow the Adaptor Plate to come to room temperature for 1 hour.
o
When the Adaptor Plate is at room temperature, centrifuge the Adaptor Plate for 3 minutes at 3000 rpm.
o
Carefully pull the seal off of the Adaptor Plate without jostling it and immediately change your gloves after removing the plate seal.
o
Transfer the entire volume of each well from the Reaction Plate (25 μL) to the corresponding well in the Adaptor Plate.
o
Thaw the Ligation Buffer to room temperature and prepare the Ligation Master Mix on ice according to the following table and mix by pipetting:
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
5
POST-PCR
Reagent
Volume (μL)
Ligation Enzyme (E) Ligation Buffer (F) Total Volume
2.5 μL 30 μL 32.5 μL
Recommended volumes for 24 libraries (μL) 63.2 μL 757.5 μL 820.7 μL
Color code Blue Black
o
Aliquot the Ligation Master Mix into clean columns of the Reagent Plate and transfer 32.5 μL into each well of the Adaptor Plate using a multichannel pipette. Mix thoroughly by pipetting.
o
Cover the Adaptor Plate with a thermal seal, centrifuge for 10 seconds and incubate the Reaction Plate in a thermal cycler with the following program:
Ligation Program Number of Cycles 1 1 1
o
Temperature 25°C 65°C 4°C
Time 10 minutes 20 minutes ∞
Safe stopping point. Products can be stored at 4°C overnight or -20°C for longer periods.
Allow AMPure XP beads to come to room temperature, vortex the tube thoroughly to mix and prepare 5 mL of 80% ethanol (4 mL EtOH + 1 mL H2O).
o
Create the Library by combining an aliquot from each pooled amplicon, now a sample-specific library, into a single 2.0 mL low bind microcentrifuge tube. I. For 16 or more samples - Calculate the amount of each sample library to pool together as a single Library of 900 μL total volume. Divide 900 μL by the number of sample libraries. This is the volume of aliquot to be taken from each sample library and pipetted into the Library. II. For fewer than 16 samples – Transfer 60 μL of each sample library into a Library.
o
Add 900 μL of AMPure XP beads to the Library tube, mix thoroughly on a vortex and centrifuge briefly. Do not allow the beads to separate. If the volume of the Library is less than 900 μL, add an equal volume of the beads to it. Always keep the bead to Library ratio to be 1:1.
o o o
Incubate the Library tube for 10 minutes at room temperature.
o
Add ~1.5 - 2 mL of freshly prepared 80% ethanol to the Library tube, leaving the tube on the magnetic stand.
o
Incubate the Library tube at room temperature for 30 seconds; afterwards, carefully remove and discard the supernatant.
o
Repeat the last two steps (wash with 80% ethanol, incubate for 30 seconds on magnetic rack and discard the supernatant).
o
Centrifuge the Library tube for 5 seconds, quickly place the tube back on the magnet and remove residual ethanol with a pipette. Do not touch the beads with the pipette.
Place the Library tube onto a magnetic stand and incubate for 10 minutes. Carefully remove and discard the supernatant from the Library tube, leaving the tube on the magnetic stand. Be careful not to disturb the beads.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
6
POST-PCR
Remove as much ethanol as possible without disturbing the beads!
o o
Air-dry the Library for 5 minutes on the magnetic stand.
o
Vortex the Library tube to resuspend the beads, and centrifuge briefly. Do not allow the beads to separate.
o o o
Incubate the Library tube at room temperature for 2 minutes.
Remove the Library tube from the magnetic stand and elute DNA by adding 31 μL molecular grade water above the beads in the tube.
Place the Library tube on the magnetic stand for 2 minutes. Transfer 31 μL from the Library tube to a new 1.5 mL low bind microcentrifuge tube.
Safe stopping point. Library can be stored at -20°C.
Step 5 – Library Size Selection
o
Bring marker K loading dye for use with the Pippin Prep (marker R2 for Blue Pippin) to room temperature.
o
Mix 10 μL marker K loading dye with the 31 μL purified Library, vortex to mix and spin down for 1 second.
o
Prepare the Pippin Prep according to manufacturer’s instructions and configure it to collect DNA fragments between 650 and 1300 bp, load 40 μL library in the sample port and run.
o
Collect the entire volume of the size selected library from the collection port of the Pippin Prep into a new 1.5 ml low bind microcentrifuge tube.
Safe stopping point. Library can be stored at -20°C.
Step 6 – Library Quantitation
o
Prepare Qubit assay tubes (500 microliter, thin-walled) for your library in duplicate and the two standards. Vortex and centrifuge the standards and the samples.
o
Add X * 199 µL from Buffer and X * 1 µL from dye to a 10 ml centrifuge tube. Vortex it for a few seconds. X = # of samples + 2 standards + 1 for pipetting loss.
o
Pipet 190 µL from the mix to the two standards' Qubit tubes. Pipet 198 µL from the mix to the samples' Qubit tubes.
o o o o
Pipet 10 µL from standard 1 to the Qubit tube and vortex it for 2 seconds. Repeat with standard 2. Pipet 2 µL from the library to their Qubit tubes and vortex for 2 seconds. Wait for 2 mins. Switch on Qubit machine and choose BR protocol. . Put standard 1 Qubit tube in and push GO. Repeat with standard 2.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
7
POST-PCR o
Put the library tube in Qubit and push GO. Then put the replicate. Determine the library concentration and dilute 10 μL of library to a 2nM concentration with molecular grade H2O in a fresh 1.5-mL low bind microcentrifuge tube.
o
Store the remaining Size Selected Library at -20° C.
Step 7 – Sequencing on Illumina’s MiSeq Quick tip – You can use a 1% PhiX spike-in as an additional control to monitor the sequencing reaction.
o o
Prepare the MiSeq instrument and the MiSeq Reagent kit v2 according to manufacturer’s instructions.
o o
Incubate the 1 nM Denatured Pooled Library at room temperature for 5 minutes.
o
Prepare a 9 pM Denatured Pooled Library by adding 550 μL of chilled HT1 and 450 μL of the 20 pM Denatured Pooled Library to a fresh 1.5 mL low bind microcentrifuge tube. Vortex and spin down for 1 second.
o
Transfer 600 μL of the 9 pM Denatured Library into the Load Samples reservoir of the MiSeq reagent cartridge.
Prepare a 1 nM Denatured Pooled Library by combining 10 μL of freshly prepared 0.2 N NaOH and 10 μL of the 2 nM Diluted Pooled Library in a new 1.5 mL low bind microcentrifuge tube. Vortex and spin down for 1 second. Prepare a 20 pM Denatured Pooled Library by adding 980 μL of chilled HT1 to the 20 μL of the 1 nM Denatured Pooled Library. Vortex and spin down for 1 second.
Step 8 – Analysis of HLA Sequencing Data
o
Analyze HLA Sequencing Data with HLA Twin according to your IT setup.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
8
Step 0 – Genomic DNA Preparation
o
Dilute all gDNAs to a concentration of 20 - 30ng/µL (minimum volume is 45 µL).
o
Thaw HLA-A, B, C, DRB1, DPB1, DQA1, DQB1 (set 1) and DQB1 (set 2) primer sets, Enhancer 1, Enhancer 2, Long-Range PCR Buffer (10X) and dNTP mix (10mM each) to room temperature.
o
Prepare the Combined Enhancer. Add 132 μL of Enhancer 2 directly into the Enhancer 1 tube, and relabel the tube as Combined Enhancer.
Step 1 – HLA Amplification Master Mix Preparation
o
Prepare a Master Mix for each HLA locus according to the following tables:
Master Mix Table for HLA-A, B, C, DRB1, DPB1 and DQA1 Reagent Primer Mix LongRange PCR Buffer (10×) dNTP Mix (10 mM each) Molecular grade H2O Total Volume
Volume/sample/locus 2 μL 2.5 μL 1.25 μL 13.85 μL 19.6 μL
Volume/24 samples/locus 51 μL 63.8 μL 31.9 μL 353.2 μL 499.9 μL
Master Mix Table for HLA-DQB1 Set 1 (Optional) and HLA-DQB1 Set 2 (Required) Reagent Primer Mix LongRange PCR Buffer (10×) dNTP Mix (10 mM each) Combined Enhancer Molecular grade H2O Total Volume
Volume/sample/locus 2 μL 2.5 μL 1.25 μL 5.6 μL 7.85 μL 19.2 μL
Volume/24 samples/locus 51 μL 63.8 μL 31.9 μL 142.8 μL 200.2 μL 489.7 μL
o
Vortex each Master Mix and spin down for 1 second. Place Master Mixes on ice.
Step 2 – HLA Class I and II Amplification
o
Remove the Taq Polymerase from storage, spin it down, and add it to each Master Mix according to the tables below, rinsing the pipette tips thoroughly by pipetting:
Taq Polymerase-Loaded Master Mix: HLA-A, B, C, DRB1, DPB1, and DQA1 Reagent Master Mix from Step 1 Taq Polymerase Total Volume
Volume/sample/locus 19.6 μL 0.4 μL 20 μL
Volume/24 samples/locus 499.9 μL 10.2 μL 510.1 μL
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
1
PRE-PCR Taq Polymerase-Loaded Master Mix: HLA-DQB1 Set 1 (Optional) and HLA-DQB1 Set 2 (Required) Reagent Master Mix from Step 1 Taq Polymerase Total Volume
Volume/sample/locus 19.2 μL 0.8 μL 20 μL
Volume/24 samples/locus 489.7 μL 20.4 μL 510.1 μL
o o
Briefly vortex and spin down all Taq Polymerase-loaded Master Mixes.
o
Add 5 μL of each diluted sample of gDNA into the designated wells of each locus-specific 96-well Amplification Plate.
o
Seal the plates with a thermal seal, centrifuge for 10 seconds, place the Class I and Class II Amplification Plates into separate thermal cyclers and run the following programs:
Aliquot 20 μL of the appropriate Taq Polymerase-loaded Master Mix for each locus into the Class I and Class II Amplification Plates.
Class I Amplification Number of Cycles 1 35 1 1
Class II Amplification
Temperature
Time
95°C 95°C 65°C 68°C 68°C 4°C
3 minutes 15 seconds 30 seconds 5 minutes 10 minutes ∞
Number of Cycles 1 35 1 1
Temperature
Time
95°C 93°C 60°C 68°C 68°C 4°C
3 minutes 15 seconds 30 seconds 9 minutes 10 minutes ∞
Safe stopping point. Amplicons can be stored at 4°C overnight or -20°C for longer periods.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
2
POST-PCR Step 3 – Amplicon Quantitation and Normalization Quick tip – A concentration of 50 ng/μL or greater is considered a successful amplification.
o o
Remove the QuantiFluor dsDNA dye system from the fridge and let it come to room temperature. Follow the dilution table below to create a serial dilution of the QuantiFluor Lambda DNA standard (100 ng/μL):
Label on tube Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Blank
Input DNA
Volume DNA (μL)
Lambda DNA Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Blank
7.5 μL 250 μL 250 μL 250 μL 250 μL 250 μL 0 μL
Volume 1x TE (μL) 492.5 μL 250 μL 250 μL 250 μL 250 μL 250 μL 250 μL
Final Conc. (ng/μL) 1.5 ng/μL 0.75 ng/μL 0.38 ng/μL 0.19 ng/μL 0.09 ng/μL 0.05 ng/μL 0 ng/μL
o
Dilute 1 μL of amplicon DNA from the Amplicon Plate into 99 μL 1× TE buffer in a 96-well optical plate. Seal the plate, vortex well and centrifuge plate for 10 seconds.
o
Prepare 1× QuantiFluor Dye working solution by mixing 0.5 μL QuantiFluor Dye (200X) + 99.5 μL 1× TE buffer per sample. Prepare sufficient 1× QuantiFluor Dye working solution so that each sample (total number of samples in Amplicon Plates) and standard (14 total) will receive a 100 µL aliquot.
o
Aliquot 100 μL of 1× QuantiFluor Dye working solution into the appropriate wells of two 96-well optical plates to prepare a Standards Quantitation Plate and an Amplicon Quantitation plate.
o
Add 100 μL of each standard, in duplicate, to individual wells in the Standards Quantitation Plate (14 wells total). Mix well by pipetting without creating bubbles. NOTE: If using a qPCR machine use qPCR machine-compatible plates and divide all volumes in half (combine 50 µL of Quantifluor working solution with 50 µL of diluted amplicons or 50 µL of standards).
o
Run the Standards Quantitation Plate on the plate fluorometer (or qPCR machine) followed by the Amplicon Quantitation Plate and calculate the concentration of DNA in the Amplicon Quantitation Plate using RFU data generated by the plate fluorometer (or qPCR machine).
o
Dilute DNA in the Amplicon Plate with molecular grade H2O so that the final concentration of DNA is approximately 67 ng/μL. • If DNA concentration is 150 ng/μL or greater: add 25 μL of H2O • If DNA concentration is 100-150 ng/μL: add 10 μL of H2O • If DNA concentration is less than 100 ng/μL: add 0 μL of H2O
o o
For each sample, combine 15 μL of the DQB1 (Set 1) and DQB1 (Set 2) into an unused well (only for SUCCESSFUL amplifications if both of DQB1 sets were used). Pool all loci for each sample into a single Amplicon Plate by combining 5 μL from each locus of a sample into a new 96-well PCR plate. Each sample should occupy a single well, with a volume of 35 μL.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
3
POST-PCR o o
Mix well by pipetting.
Aliquot 4 μL of ExoSAP-IT Express (Affymetrix) into each amplicon in the Class I and Class II Amplification Plates using a multichannel pipette and mix by pipetting. Seal the Amplification Plates with a thermal seal, centrifuge for 10 seconds, place them into a thermal cycler and run the following program: ExoSAP-IT Express Program Number of Cycles 1 1 1
Temperature 37°C 80°C 4°C
Time 4 minutes 1 minutes ∞
Safe stopping point. Amplicons can be stored at 4°C overnight or -20°C for longer periods.
Step 4 – Library Preparation
The reagents used during library preparation are very viscous and particular attention is required while pipetting them. *IMPORTANT* - Use PCR Freezer Blocks to setup the Fragmentation reactions. It’s very important to keep all the reagents cold at all times and to pre-heat the thermal cycler prior to putting the plate on it.
o o o
Thaw the Fragmentation Buffer to room temperature and keep it on ice. Vortex the Fragmentation enzyme thoroughly. Prepare the Fragmentation Master Mix on ice according to the following table and vortex the Fragmentation Mix to mix:
Reagent Fragmentation Enzyme (A) Fragmentation Buffer (B) Total volume
Volume per library (μL) 2 μL 2 μL 4 μL
Recommended volumes per 24 libraries (μL) 55.2 μL 55.2 μL 110.4 μL
Color code Yellow Red
o
Place a 96-well Reagent Plate on a PCR cooler block, aliquot the Fragmentation Master Mix in a clean column and spin for 10 seconds.
o
Place a 96-well Reaction Plate on a second PCR cooler block and aliquot 4 μL of Fragmentation Master Mix from the Reagent Plate into each well of the Reaction Plate that will be used with a multichannel pipette.
o
Replace the Reagent Plate with the Amplicons Plate on the PCR cooler block and transfer 16 μL of each amplicon from the Amplicons Plate to the corresponding well on the Reaction Plate using a multichannel pipette. Mix by pipetting.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
4
POST-PCR o
Cover the Reaction Plate with a thermal seal, centrifuge for 10 seconds and incubate the Reaction Plate in a thermal cycler with the following program: Fragmentation Program Number of Cycles 1 1 1
Time 10 minutes 15 minutes ∞
Safe stopping point. Products can be stored at 4°C overnight or -20°C for longer periods.
o o
Temperature 37° C 70° C 4° C
Thaw the End Repair Buffer to room temperature and keep it on ice. Prepare the End Repair Master Mix on ice according to the following table and mix by pipetting:
Reagent
Volume per library (μL)
Molecular grade H2O End Repair Enzyme (C) End Repair Buffer (D) Total volume
1.25 μL 1.25 μL 2.5 μL 5 μL
Recommended volumes for 24 libraries (μL) 34.8 μL 34.8 μL 69.6 μL 139.2 μL
Color code Green Orange
o
Aliquot the End Repair Master Mix in a clean column of the Reagent Plate and transfer 5 μL into each well of the Reaction Plate using a multichannel pipette. Mix thoroughly by pipetting.
o
Cover the Reaction Plate with a thermal seal, centrifuge for 10 seconds and incubate the Reaction Plate in a thermal cycler with the following program:
End Repair Program Number of Cycles 1 1 1
Temperature 20°C 65°C 4°C
Time 30 minutes 30 minutes ∞
Safe stopping point. Products can be stored at 4°C overnight or -20°C for longer periods.
o
Remove Indexed Adaptor Plate from -20°C and allow the Adaptor Plate to come to room temperature for 1 hour.
o
When the Adaptor Plate is at room temperature, centrifuge the Adaptor Plate for 3 minutes at 3000 rpm.
o
Carefully pull the seal off of the Adaptor Plate without jostling it and immediately change your gloves after removing the plate seal.
o
Transfer the entire volume of each well from the Reaction Plate (25 μL) to the corresponding well in the Adaptor Plate.
o
Thaw the Ligation Buffer to room temperature and prepare the Ligation Master Mix on ice according to the following table and mix by pipetting:
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
5
POST-PCR
Reagent
Volume (μL)
Ligation Enzyme (E) Ligation Buffer (F) Total Volume
2.5 μL 30 μL 32.5 μL
Recommended volumes for 24 libraries (μL) 63.2 μL 757.5 μL 820.7 μL
Color code Blue Black
o
Aliquot the Ligation Master Mix into clean columns of the Reagent Plate and transfer 32.5 μL into each well of the Adaptor Plate using a multichannel pipette. Mix thoroughly by pipetting.
o
Cover the Adaptor Plate with a thermal seal, centrifuge for 10 seconds and incubate the Reaction Plate in a thermal cycler with the following program:
Ligation Program Number of Cycles 1 1 1
o
Temperature 25°C 65°C 4°C
Time 10 minutes 20 minutes ∞
Safe stopping point. Products can be stored at 4°C overnight or -20°C for longer periods.
Allow AMPure XP beads to come to room temperature, vortex the tube thoroughly to mix and prepare 5 mL of 80% ethanol (4 mL EtOH + 1 mL H2O).
o
Create the Library by combining an aliquot from each pooled amplicon, now a sample-specific library, into a single 2.0 mL low bind microcentrifuge tube. I. For 16 or more samples - Calculate the amount of each sample library to pool together as a single Library of 900 μL total volume. Divide 900 μL by the number of sample libraries. This is the volume of aliquot to be taken from each sample library and pipetted into the Library. II. For fewer than 16 samples – Transfer 60 μL of each sample library into a Library.
o
Add 900 μL of AMPure XP beads to the Library tube, mix thoroughly on a vortex and centrifuge briefly. Do not allow the beads to separate. If the volume of the Library is less than 900 μL, add an equal volume of the beads to it. Always keep the bead to Library ratio to be 1:1.
o o o
Incubate the Library tube for 10 minutes at room temperature.
o
Add ~1.5 - 2 mL of freshly prepared 80% ethanol to the Library tube, leaving the tube on the magnetic stand.
o
Incubate the Library tube at room temperature for 30 seconds; afterwards, carefully remove and discard the supernatant.
o
Repeat the last two steps (wash with 80% ethanol, incubate for 30 seconds on magnetic rack and discard the supernatant).
o
Centrifuge the Library tube for 5 seconds, quickly place the tube back on the magnet and remove residual ethanol with a pipette. Do not touch the beads with the pipette.
Place the Library tube onto a magnetic stand and incubate for 10 minutes. Carefully remove and discard the supernatant from the Library tube, leaving the tube on the magnetic stand. Be careful not to disturb the beads.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
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POST-PCR
Remove as much ethanol as possible without disturbing the beads!
o o
Air-dry the Library for 5 minutes on the magnetic stand.
o
Vortex the Library tube to resuspend the beads, and centrifuge briefly. Do not allow the beads to separate.
o o o
Incubate the Library tube at room temperature for 2 minutes.
Remove the Library tube from the magnetic stand and elute DNA by adding 31 μL molecular grade water above the beads in the tube.
Place the Library tube on the magnetic stand for 2 minutes. Transfer 31 μL from the Library tube to a new 1.5 mL low bind microcentrifuge tube.
Safe stopping point. Library can be stored at -20°C.
Step 5 – Library Size Selection
o
Bring marker K loading dye for use with the Pippin Prep (marker R2 for Blue Pippin) to room temperature.
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Mix 10 μL marker K loading dye with the 31 μL purified Library, vortex to mix and spin down for 1 second.
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Prepare the Pippin Prep according to manufacturer’s instructions and configure it to collect DNA fragments between 650 and 1300 bp, load 40 μL library in the sample port and run.
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Collect the entire volume of the size selected library from the collection port of the Pippin Prep into a new 1.5 ml low bind microcentrifuge tube.
Safe stopping point. Library can be stored at -20°C.
Step 6 – Library Quantitation
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Prepare Qubit assay tubes (500 microliter, thin-walled) for your library in duplicate and the two standards. Vortex and centrifuge the standards and the samples.
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Add X * 199 µL from Buffer and X * 1 µL from dye to a 10 ml centrifuge tube. Vortex it for a few seconds. X = # of samples + 2 standards + 1 for pipetting loss.
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Pipet 190 µL from the mix to the two standards' Qubit tubes. Pipet 198 µL from the mix to the samples' Qubit tubes.
o o o o
Pipet 10 µL from standard 1 to the Qubit tube and vortex it for 2 seconds. Repeat with standard 2. Pipet 2 µL from the library to their Qubit tubes and vortex for 2 seconds. Wait for 2 mins. Switch on Qubit machine and choose BR protocol. . Put standard 1 Qubit tube in and push GO. Repeat with standard 2.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
7
POST-PCR o
Put the library tube in Qubit and push GO. Then put the replicate. Determine the library concentration and dilute 10 μL of library to a 2nM concentration with molecular grade H2O in a fresh 1.5-mL low bind microcentrifuge tube.
o
Store the remaining Size Selected Library at -20° C.
Step 7 – Sequencing on Illumina’s MiSeq Quick tip – You can use a 1% PhiX spike-in as an additional control to monitor the sequencing reaction.
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Prepare the MiSeq instrument and the MiSeq Reagent kit v2 according to manufacturer’s instructions.
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Incubate the 1 nM Denatured Pooled Library at room temperature for 5 minutes.
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Prepare a 9 pM Denatured Pooled Library by adding 550 μL of chilled HT1 and 450 μL of the 20 pM Denatured Pooled Library to a fresh 1.5 mL low bind microcentrifuge tube. Vortex and spin down for 1 second.
o
Transfer 600 μL of the 9 pM Denatured Library into the Load Samples reservoir of the MiSeq reagent cartridge.
Prepare a 1 nM Denatured Pooled Library by combining 10 μL of freshly prepared 0.2 N NaOH and 10 μL of the 2 nM Diluted Pooled Library in a new 1.5 mL low bind microcentrifuge tube. Vortex and spin down for 1 second. Prepare a 20 pM Denatured Pooled Library by adding 980 μL of chilled HT1 to the 20 μL of the 1 nM Denatured Pooled Library. Vortex and spin down for 1 second.
Step 8 – Analysis of HLA Sequencing Data
o
Analyze HLA Sequencing Data with HLA Twin according to your IT setup.
Omixon Holotype HLA 24/7 RUO Quick Protocol Guide v2.0 Copyright© 2017, Omixon Biocomputing Ltd. Confidential & Proprietary
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