Oct 16, 2024
Version 2
HEP-TILE: HBV whole genome sequencing (nanopore protocol) V.2
Version 1 is forked from ARTIC SARS-CoV-2 sequencing protocol v4 (LSK114)
- 1University of Oxford;
- 2University of Birmingham;
- 3The Francis Crick Institute;
- 4ARTIC Network
Protocol Citation: Sheila Lumley, Josh Quick, Philippa Matthews, Chris Kent 2024. HEP-TILE: HBV whole genome sequencing (nanopore protocol). protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82bedl2w/v2Version created by Sheila Lumley
Manuscript citation:
Whole genome sequencing of hepatitis B virus (HBV) using tiled amplicon (HEP-TILE) and probe-based enrichment on Illumina and Nanopore platforms. Lumley et al. 2024 https://www.medrxiv.org/content/10.1101/2024.09.11.24313306v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 09, 2024
Last Modified: October 16, 2024
Protocol Integer ID: 110012
Keywords: hepatitis b virus, whole genome sequencing, HBV, tiled amplicon, nanopore sequencing
Funders Acknowledgement:
Wellcome trust
Grant ID: 102176/B/13/Z
Abstract
This protocol describes the HEP-TILE tiled amplicon protocol for whole genome sequencing of Hepatitis B virus (HBV) on the nanopore MinION.
We developed a pan-genotypic (genotypes A-J) HBV scheme using an early version of PrimalScheme3, a web-based primer design tool for developing multiplex primer schemes. PrimalScheme3 is a successor to PrimalScheme, with a number of changes made to enable us to generate an overlapping (tiled) amplicon scheme which covered the circular HBV genome, utilising a number of discrete primers at each position to handle intraspecies diversity.
Primer sequences
The amplicons can also be fragmented and sequenced on Illumina platforms.
Recommended extraction protocol
We use the QIAamp minelute virus spin kit with carrier RNA. For samples with VL >5log IU/ml we extract from 200ul of sample using the manufacturers protocol, for samples with VL <5log IU/ml we extract from 400ul sample doubling up the protease, AL and ethanol.
Materials
| A | B | C | |
| Component | Supplier | Part number | |
| HEP-TILE primers hbv/600/v2.1.0 | IDT | See links below | |
| Q5 Hot Start High-Fidelity 2X Master Mix | NEB | M0494 | |
| Nuclease-free water (100 mL) | NEB | B1500 | |
| SPRI-select beads | Beckman | B23318 | |
| Ethanol | |||
| NEBNext Ultra II End Repair/dA-tailing module | NEB | E7546 | |
| Blunt/TA Ligase Master Mix | NEB | M0367 | |
| NEBNext Quick Ligation Module | NEB | E6056S | |
| Native Barcoding Kit 24 V14 or | ONT | SQK-NBD114.24 | |
| Native Barcoding Kit 96 V14 | ONT | SQK-NBD114.96 | |
| Native Barcoding Auxiliary Kit V14 (optional) | ONT | EXP-NBA114 | |
| Short Fragment Buffer Expansion Kit | ONT | EXP-SFB001 | |
| Flow Cell Priming Kit (optional) | ONT | EXP-FLP004 | |
| Flow Cell Wash Kit (optional) | ONT | EXP-WSH004 | |
| R10.4.1 flow cells | ONT | FLO-MIN114 | |
| Bovine serum albumin (50mg/ml) | Invitrogen | ||
| AMPure XP beads | Beckman | A63881 | |
| Qubit dsDNA HS Assay Kit | Thermo | Q32854 |
Order oligos listed here individually:
Before start
Prepare between 11 and 95 DNA samples plus 1 negative control using this protocol.
Sample preparation
Sample preparation
Prepare between 11 and 94 DNA samples plus 1 negative control of nuclease-free water per library (a 2nd negative control is added at the start of library prep).
Batch samples by viral load, processing samples above/below 5 log10 IU/ml separately.
If previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on ice at all times.
Note
A positive control can also be included which may be a the NIBSC HBV control or clinical sample. This can help monitor run performance.
Primer pool preparation
Primer pool preparation
If making up primer pools from individual oligos fully resuspend lyophilised oligos in 1xTE to a concentration of 100 micromolar (µM) , vortex thoroughly and spin down.
Sort all odd regions primers into one or more tube racks. Add 5 µL of each odd region primer to a 1.5 mL Eppendorf tube labelled "Pool 1 (100 micromolar (µM) )". Repeat the process for all even region primers for Pool 2. These are your 100 micromolar (µM) stocks of each primer pool.
Note
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
Note
For more information see Figure 2 in;
Quick, J. et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc 12, 1261–1276 (2017). https://doi.org/10.1038/nprot.2017.066
Dilute 100 micromolar (µM) pools 1:10 in molecular grade water, to generate 10 micromolar (µM) primer stocks.
Note
Primers are used at a final concentration of 15 nanomolar (nM) per primer. In this case hbv/600/v2.1.0 pools have 69 primers in pool 1 and 63 primers in pool 2. so the requirement is ~2.5 µL primer pool (10 micromolar (µM) ) per 25 µL reaction.
Note
Make up multiple 100 µL aliquots of 10 micromolar (µM) primer dilutions and freeze them in case of degradation or contamination.
Multiplex PCR
Multiplex PCR
Set up the two PCR reactions per sample as follows in strip-tubes or plates. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Samples > 5log10 IU/ml
| A | B | C | |
| Component | Reaction 1 | Reaction 2 | |
| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µL | 12.5 µL | |
| V3 Pool 1 (10µM) | 2.5 µL | 0 µL | |
| V3 Pool 2 (10µM) | 0 µL | 2.5 µL | |
| Nuclease-free water | 7.5 µL | 7.5 µL | |
| (Sample) | (2.5ul) | (2.5ul) | |
| Total | 25 µL | 25 µL |
Samples < 5log10 IU/ml
| A | B | C | |
| Component | Reaction 1 | Reaction 2 | |
| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µL | 12.5 µL | |
| V3 Pool 1 (10µM) | 2.5 µL | 0 µL | |
| V3 Pool 2 (10µM) | 0 µL | 2.5 µL | |
| Nuclease-free water | 2.5 µL | 2.5 µL | |
| (Sample) | (7ul) | (7ul) | |
| Total | 25 µL | 25 µL |
Note
Up to 7 µL DNA can be added to each PCR reaction (in place of nuclease-free water) to improve amplification of low titre samples.
> 5 log10 IU/ml use 2.5ul
< 5 log10 IU/ml use 7ul
PCR reactions can be performed in duplicate for low viral load samples e.g. < 3 log10 IU/ml
Note
To prevent pre-PCR contamination the mastermix for each pool should be made up in the mastermix cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use and aliquoted into PCR strip-tubes/plate
Add DNA to each of the PCR reactions (volume determined by VL of sample, see step 5), gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Note
DNA should be added in the DNA/sample addition cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
Set-up the following program on the thermal cycler, use 35 cycles if VL > 5 log10 IU/ml, use 40 cycles if VL < 5 log10 IU/ml.
Step Temperature Time Cycles
Heat Activation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:15 35 or 40
Annealing 65 °C 00:05:00 35 or 40
Hold 4 °C Indefinite 1
Bead clean up
Bead clean up
Label strip-tubes/plate and combine the following volumes of each PCR reaction for 25 µL each sample:
| A | B | |
| Component | Volume | |
| Pool 1 PCR reaction | 25 μL | |
| Pool 2 PCR reaction | 25 µL | |
| Total | 50 µL |
Note
Amplicons should be added in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
Add 40 µL SPRI beads to 50 µL pooled PCR product for an 0.8x clean up.
Gently mix and incubate for 00:05:00 at room temperature
5m
Place on a magnetic rack and incubate for 00:02:00 or until the beads have pelletted and supernatant is completely clear. Remove and discard the supernatant.
2m
Perform an ethanol wash: add 100 µL of room temperature 70% ethanol to bathe the pellet, carefully remove and discard ethanol being careful not to touch the bead pellet.
Repeat the ethanol wash (step 9.2)
Pulse centrifuge to collect and remove residual ethanol, remove excess with a P10 pipette, it is very important all is removed.
With the tube lid open, air dry for 1-2 minutes, or until the pellet loses its shine. Do not over-dry.
Re-suspend pellet in 20 µL H20, mix gently and incubate for 2 minutes at room temperature
Place on magetic rack until clear, transfer the supernatant to clean labelled tubes, do not disturb the beads.
Quantify using a Qubit or Quantus.
End preparation
End preparation
In a new PCR strip-tube/plate set up the following reaction for each sample, normalising to 80ng amplicon DNA input per sample. Add an additional reagent-only negative control at this step.
| A | B | |
| Component | Volume | |
| Clean PCR product from previous step | Up to 8.3 ul | |
| Ultra II end prep reaction buffer | 1.2ul | |
| Ultra II end prep enzyme mix | 0.5ul | |
| Nuclease-free water | to a total of 10ul | |
| Total | 10ul |
Note
Make a master mix of end-preparation reagents and nuclease-free water and aliquot into strip-tube/plate to improve reproducability.
Incubate at room temperature for 00:15:00
Incubate at 65 °C for 00:15:00
Incubate on ice for 00:01:00
31m
Native barcoding
Native barcoding
In a new PCR strip-tube/plate set up the following reaction for each sample:
| A | B | |
| Component | Volume | |
| End-preparation reaction mixture | 1 ul | |
| NBXX barcode | 1.25 ul | |
| Blunt/TA Ligase master mix | 5 ul | |
| Nuclease-free water | 2.75 ul | |
| Total | 10 ul |
Note
Use one native barcode per sample. Use 12 or more barcodes per library or there will be insufficient total material to achieve good yields.
Incubate at room temperature for 00:20:00
20m
Add EDTA and mix thoroughly
| A | B | |
| EDTA cap colour | Volume per well | |
| For clear cap EDTA | 1ul | |
| For blue cap EDTA | 2ul |
Note
NB. EDTA concentration varies by cap colour supplied by ONT
Note
Alternatively a 65C heat step can be used to inactivate the ligase - see ONT version of protocol.
In a new 1.5ml Eppendorf tube pull all one-pot barcoding reactions together.
Note
If processing 12-24 samples pool all 10 µL from each native barcoding reaction.
if processing 48 samples pool 5 µL from each native barcoding reaction.
If processing 96 samples pool 2.5 µL from each native barcoding reaction so as not to exceed a pool volume of 240 µL which would make the clean-up volume too large.
Determine volume of pooled barcoded samples, add an equal volume of water
Add ampure beads (supplied with ONT kit) for a 1.5x clean up. If very large volume split between 2 tubes
Example:
| A | B | |
| Component | Example volumes | |
| Pooled barcoded samples | 240 ul | |
| Water | 240 ul | |
| Beads | 720 ul |
Note
NB. 0.4x clean up in ONT protocol was leading to loss of whole library.
Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 00:05:00 at room temperature.
5m
Place on magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
2m
Add 250 µL SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
Repeat step 11.9 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
Note
You do not need to allow to air dry with SFB washes.
Add 200 µL of room-temperature 70 % ethanol to bathe the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Note
Only perform 1x 70% ethanol wash
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open incubate for 00:03:00 or until the pellet loses it's shine (very large pellets will take longer, NB if the pellet dries completely it will crack and become difficult to resuspend).
3m
00:02:00 Re-suspend pellet in 31 µL nuclease free water, mix gently by either flicking or pipetting and incubate for 00:02:00 . If beads were split between two eppendorfs then combine at this point to elute in a total of 31ul.
4m
Place on magnet and transfer sample to a clean 1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.
Quantify 1 µL of the barcoded amplicons using a fluorometer such as a Qubit or Quantus. Concentration will vary depending on number and Ct of samples and but you need about 30 ng total at this stage to achieve maximum run yield.
Adaptor ligation
Adaptor ligation
Set up the following Native Adapter (NA) ligation and clean-up with SFB.
In a new 1.5ml eppendorf or PCR tube set up the following adapter ligation reaction
| A | B | |
| Component | Volume | |
| Barcoded amplicon pool | 30 ul | |
| NEBNext quick ligation reaction buffer (5X) | 10 ul | |
| Adaptor mix (NA) | 5 ul | |
| Quick T4 DNA ligase | 5 ul | |
| Total | 50 ul |
Incubate at room temperature for 00:20:00
20m
Perform a bead clean up.
Add 50ul ampure beads (supplied with ONT kit) to the sample tube. Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 00:05:00 at room temperature.
Place on magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add 250 µL SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
Repeat step 15.3 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
Note
You do not need to allow to air dry with SFB washes.
Re-suspend pellet in 15 µL EB (ONT), mix gently by either flicking or pipetting and incubate for 00:02:00
2m
Place on magnet and transfer sample to a clean 1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.
Quantify the final library using a fluorometer such as a Qubit or Quatus.
Note
Concentration will vary depending on number and Ct of samples, expected quantification between 2-9 ng/ul, 15 ng final library is usually required to acheive maximum run yield.
Note
Final library can be now be stored at 4 °C for up to a week if needed otherwise proceed directly to MinION sequencing.
Flow cell check and priming
Flow cell check and priming
Refer to ONT documentation for images explaining how to check, prime and load flow cell.
Complete a flow cell check - open Minknow user interface, plug in MinION to laptop, open the MinION lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact. Click flow cell check on Minknow interface.
To prepare the flow cell priming mix, combine the following reagents in the FCF tube and mix by inverting the tube and pipette mix at room temperature:
| A | B | |
| Component | Volume | |
| Flow cell flush (FCF) | 1170 ul | |
| Bovine serum albumin (BSA) at 50mg/ml | 5 ul | |
| Flow cell tether (FCT) | 30 ul | |
| Final volume in FCF tube | 1205 ul |
Slide the flow cell priming port cover clockwise to open the priming port
After opening the priming port, set a P1000 pipette to 200ul, insert the tip into the priming port, turn the wheel until the dial shows 220-230ul, to draw back 20-30ul or until you can see a small volume of buffer entering the pipette tip.
Visually check that there is continuous buffer from the priming port across the sensor array.
Load 800 µL of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time prepare the library for loading by following the steps below.
MinION sequencing
MinION sequencing
Thoroughly mix the contents of the library beads by pipetting, it is vial that they are mixed immediately before use.
Note
From experience we have successfully loaded 20ng library
In a new 1.5ml Eppendorf DNA LoBind tube, prepare the library for loading as follows (NB load the library onto flow cell immediately after adding SB and LIB because the fuel in the buffer will start to be consumed by the adapter):
| A | B | |
| Reagent | Volume | |
| Sequencing buffer (SB) | 37.5 ul | |
| Library beads (LIB) mixed immediately before use | 25.5 ul | |
| DNA library + H2O | 12 ul | |
| Total | 75 ul |
Complete flow cell priming: lift the spot on sample port cover, load 200 µL of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles
Mix the prepared library gently by pipetting just prior to loading
Add half of the library (~37.5ul) to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next. Resuspend beads/library and add the 2nd half in a dropwise fashion.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
Place the light shield onto the flow cell. And close the device lid.
Start the sequencing run using MinKnow.
Note
If using live basecalling, turn on double-ended barcoding in the basecalling settings.
Protocol references
Pre-print coming soon: Whole genome sequencing of hepatitis B virus (HBV) using tiled amplicon (HEP-TILE) and probe-based enrichment on Illumina and Nanopore platforms. Lumley et al. 2024