TITLE: Complete Hepatitis B Virus Sequencing using an ONT-Based Next-Generation Sequencing Protocol
AUTHORS: Wonderful T. CHOGA, yeshnee.m, Lucious B. Chabuka, Tongai Maponga, Derek Tshiabuila, James Emmanuel San, Houriiyah Tegally, Monika Moir, Sureshnee Pillay, Richard Lessells, Cheryl Baxter, Jennifer Giandhari, Eduan Wilkinson, Tulio De Oliveira

[DESCRIPTION]
This protocol outlines the process of generating complete Hepatitis B virus (HBV) primers from DNA extracts for next-generation sequencing using Oxford Nanopore Technology (ONT). Specifically, we have designed pan-genotypic tiling primers to cover the entire HBV genome for sequencing. The hands-on time required for a batch of 48 samples is minimal, approximately 1 hour and 30 minutes. This protocol is straightforward and can be easily adapted in settings where the ONT protocol for SARS-CoV-2 has been implemented. We provide detailed instructions for PCR amplification using HBV-specific tiling primers, sample pooling, library construction using the Rapid Barcoding Kit (SQK-RBK110.96), quantification using Qubit, and subsequent sequencing on the GridION platform.

The Rapid barcoding protocol for up-to 96 samples "PCR tiling of SARS-CoV-2 virus with rapid barcoding (SQK-RBK110.96)" https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021. 

This protocol describes a modified version of the 1200bp amplicon "midnight" primer set for nCoV-2019 (SARS-CoV-2) amplification, utilizing the Nanopore Rapid kit for MinION. The modification was developed by Nikki Freed. https://dx.doi.org/10.17504/protocols.io.bwyppfvn and the original publication is found here : https://doi.org/10.1093/biomethods/bpaa014. 

Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.

Primer sequences are here (and listed directly in the protocol)  :  


[STEPS]
SECTION: Quantification of DNA using Qubit
1. Prepare the two standards calibrate the Qubit Fluorometer using Qubit dsDNA HS Assay kit Thermo Fisher Scientific (Qubit® dsDNA HS Reagent). 
SECTION: Quantification of DNA using Qubit
1.2.  For standards  [STD]: Aliquot 190 µL of Qubit® dsDNA HS Reagent working solution to each 500 µL thin-walled polypropylene tubes . 

For sample tubes: Aliquot 199 µL of Qubit® dsDNA HS Reagent  working solution. 

 

1.5. After calibrating the machine proceed to measuring DNA for samples. 

First, aliquot 199 µLof Qubit® dsDNA HS Reagent into a 500 µL thin-walled polypropylene tubes.  

Add1 µLof the specific sample to each well containing the pre-allocated 190 µL of sample.

Pulse vortex for 2 minseconds to mix thoroughly without producing bubbles. 

Incubate at room temperature for 2 min. Measure the concentation using Qubit Fluorometer.


Allow all tubes to incubate at room temperature for 2 minutes, then proceed to “Read standards and samples”.

** Do not delay (exceed) 2 min; the Qubit® dsDNA HS Reagent are light sensitive.**
 

SECTION: Quantification of DNA using Qubit
1.3. Add 10 µL of STD 1 to the aliquoted 190 µL. 


Mix each tube vigorously by vortexing for 3–5 seconds without creating bubbles. 


Incubate at room temperature for2 min prior to measuring the concentation using Qubit Fluorometer.  ** 

 

SECTION: Quantification of DNA using Qubit
1.4. After calibrating the machine with STD 1  & 2 proceed to measuring DNA for samples. 

First, aliquot 199 µLof Qubit® dsDNA HS Reagent into a 500 µL thin-walled polypropylene tubes.  

Add1 µLof the specific sample to each well containing the pre-allocated 190 µL of sample.

Mix each tube vigorously by vortexing for 3–5 seconds without creating bubbles. 

Incubate at room temperature for 2 min. Measure the concentation using Qubit Fluorometer.

 

SECTION: Primer design (Pan-genotypic/ universal tiling primers)
2. 1. Download the HBV sequences of genotypes A-J were obtained from GenBank (n>8000) (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/). 

2. Design a consensus sequence based on 50% threshold per each genotype. Annotate the conserved and parsimony informative sites.

3. Use Primal scheme (https://primalscheme.com) to design the primers and the Amplicon-size was set at 1200.

4. Compare the variable regions and select the putatively universal primers. 

5. To avoid any primer dropouts,  add the other variable primers. These can be used to spike the master mix during PCR. 

   Primer NameSequencePositionsSC_1_LEFTTTC CAC CAA GCT CTG CAA GATC11 - 32SC_1_RIGHTAGAGGAATATGATAAAACGCCGCA384-407SC_2_LEFTCATCATCATCAT CACCA CCTCC325-346SC_2_RIGHTAAAGCCCTACGAACCACTGAAC692-713SC_3_LEFTAAATACCTATGGGAGTGGGCCT632-653SC_3_RIGHTTTGTGTAAATGGAGCGGCAAAG1 655-1 676SC_4_LEFTAGAAAACTTCCTGTTAACAGACCTATTG949-976SC_4_RIGHTGGACGACAGAATTATCAGTCCCG1 326-1 348SC_5_LEFTTCCATACTGCGGAACTCCTAGC1 265-1 286SC_5_RIGHTTGTAAGACCTTGGGCAGGATTTG1 632-1 654SC_6_LEFTCTTCTCATCTGCCGGTCCGTGT1 559-1580SC_6_RIGHTAGA AGT CAG AAG GCA AAC GAGA1 947-1 970SC_7_LEFTGGCTTTGGGGCATGGACATT1 890-1 909SC_7_RIGHTATCCACACTCCGAAAGAGACCA2 256-2 277SC_8_LEFTGACAACTATTGTGGTTTCATATTTCT2 193-2 218SC_8_RIGHTTTGTTGACACCTATTAATAATGTCCTCA2 576-2 594SC_9_LEFTTGGGCTTTATTCCTCTACTGTCCC2 492-2 515SC_9_RIGHTGGGAACAGAAAGATTCGTCCCC2 889-2 910SC_10_LEFTTTGCGGGTCACCATATTCTTGG2 816-2 837SC_10_RIGHTGGCCTGAGGATGACTGTCTCTT3 189-3 210

SECTION: Reconstitution of Primer Pools
3. To ensure proper primer dilution and pooling, follow these steps in a clean master-mix hood start by decontaminating of the working area (PCR hood/cabinet ).

1. Prior to use, decontaminate the master-mix hood using 10% bleach and 70% ethanol.

2. Sterilize the master-mix hood by exposing it to ultraviolet (UV) light for15 min.

SECTION: Reconstitution of Primer Pools
3.1. Depending on the nature of the primers (lyophilised/or solution); if required, re-suspend lyophilised primers at a concentration of 100 µM each using nuclease-free water.

**Adhere to the primer reconstitution instructions provided by the supplier or manufacturer.**
SECTION: Reconstitution of Primer Pools
3.2. 1. To create a 100 µM stock of the primer pool for Pool 1, combine 5 µL of Pool 1 with 485 µL of  nuclease-free water in a labeled 1.5 mL Eppendorf tube called "Pool 1 (100 μM)". This will yield a total volume of 490 µL, resulting in a 100 µM concentration of the primer pool stock.

2. Repeat the above procedure to create 100 M of primer pool for Pool 2.
 

SECTION: Reconstitution of Primer Pools
3.3. For each 100μM primer pool (1 & 2), dilute 1:10 in molecular grade water, to generate 10 µM primer stocks. Make several aliquotes for each primer pool in case of degradation or contamination. 
 


SECTION: Tiling Polymerase chain reaction (PCR)
4. To ensure contamination free master-mixes start by decontaminating all the working area in the clean room including workbench and the master mix hood. 

#*1. Decontaminate the master-mix hood using 10% bleach and 70% ethanol.

#*2. Sterilize the mastermix hood by exposing it to ultraviolet (UV) light for 15 min.
SECTION: Tiling Polymerase chain reaction (PCR)
4.1. Each sample requires two PCR reactions (1 for each primer pool, to be combined later). 

1. Arrange the PCR reactions for each sample in strip-tubes or plates according to the following instructions. 

2. Mix the following components in a labeled 1.5ml eppendorf tube. Combine other reagents/components except the template as master-mix and divide into aliquots before adding DNA. 

3. Mix gently by pipetting and briefly spin the tube to ensure the liquid collects at the bottom.
 
ComponentPCR 1PCR 2Q5 2x Master Mix12.5 μL12.5 μLPrimer pool 1 (10µM)1 μL0 μLPrimer pool 2 (10µM)0 µL1 µLNuclease-free water8.5 μL8.5 μLcDNA3 µL3 µLTotal Volume25µL25µL

SECTION: Tiling Polymerase chain reaction (PCR)
4.2.  In clean MasterMix cabinet:  

1. Add 12.5 µL 5X Q5 Reaction Buffer to a labeled 1.5ml eppendorf tube. 

2. Add1.1 µL Primer Pool 1 or 2 (10μM) to the 1.5ml Eppendorf tube containing 12.55 µL 5X Q5 Reaction Buffer.


3. Add8.9 µL of Nuclease free-water to the 1.5ml eppendorf mixture. The total volume should now be 25 µL

4. Aliquot the 22.5 µL of master-mix in labelled PCR strip tubes and transfer the master-mixes to the decontaminated#* extraction hood.
SECTION: Tiling Polymerase chain reaction (PCR)
4.3. In the extraction and sample addition cabinet:  

Add 2.5 µL of DNA template into the master-mixes, both pool 1 and 2. After adding; mix well by pipetting. 

- Add 2.5 µL each DNA sample to a tube containing 22.5 µL Pool1. 
- Add 2.5 µL each DNA sample to a tube containing 22.5 µL Pool 2. 

2. Carefully mix the contents by pipetting in a gentle manner, and pulse centrifuge the tubes to collect the contents at the bottom of the tube.

 

SECTION: Tiling Polymerase chain reaction (PCR)
4.4. Incubate both PCR reactions in a thermocycler with the following settings:
 
Heat Activation98°C30 secondsDenaturation95°C15 secondsAnnealing65°C5 minutesRepeat denaturation and annealing for a total of 25-35 cyclesHold4°C∞
 
 

SECTION: Rapid barocoding using the SLK RBK109
9.6. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: Rapid barocoding using the SLK RBK109
9.7. Add 200 µL of freshly prepared 70% ethanol (at room temperature) to the pellet. Take caution to avoid touching the bead pellet. Remove the ethanol carefully and discard it.
SECTION: Rapid barocoding using the SLK RBK109
9.5. Place on magnetic rack and incubate for or until the beads have pelleted and the supernatant is completely clear.
SECTION: Rapid barocoding using the SLK RBK109
9.8. Repeat Step above. 
SECTION: Rapid barocoding using the SLK RBK109
9.9. Centrifuge the tube briefly in pulses to ensure that all the liquid gathers at the bottom. Subsequently, using a P10 pipette, cautiously extract as much residual ethanol as possible from the tube.
SECTION: Rapid barocoding using the SLK RBK109
9.10. Leave the tube lid open and incubate for 1 minute or until the pellet loses its shine. 

NB*It is important to note that if the pellet dries completely, it may crack and become challenging to resuspend.
SECTION: Rapid barocoding using the SLK RBK109
9.11. Resuspend pellet in 30 µLElution Buffer (EB), mix gently by either flicking or pipetting and incubate for 00:02:00.
SECTION: Rapid barocoding using the SLK RBK109
9.12. Place on magnetic stand and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
SECTION: Rapid barocoding using the SLK RBK109
9.13. Measure the concentration of samples using Qubit. (See Section 1).
SECTION: Visualising the size of amplicons using the LabChip Fragment Analyzer.
6. "To add the lab chip preparation protocol"
SECTION: Pooling and PCR quantification
5. Label a  1.5 mL Eppendorf tube for each sample.

Transfer and merge all the components from the "Pool 1" and "Pool 2" PCR reactions of each biological sample into a single 1.5 mL Eppendorf tube, ensuring that all the contents are from sample. ** Avoid mixing samples** 

Component                                            Volume
Pool 1 PCR reaction                         10 µL
Pool 2 PCR reaction                         10 µL
Total                                                      20 µL

 

SECTION: Rapid barocoding using the SLK RBK109
9.3. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Rapid barocoding using the SLK RBK109
9.4. Incubate for 00:05:00 at Room temperature. 
SECTION: Pooling and PCR quantification
5.1. To quantify the DNA, it is recommended to use a Qubit or any other suitable method. Nanodrop is not recommended for this purpose. However, if you do not have access to a Qubit or prefer to save time and costs, you may choose to omit this quantification step.

See  

SECTION: Rapid barcoding using the RBK110.96 kit for 96 samples
7. To accommodate multiple samples on the same flow cell, barcoding can be employed. Using the RBK110.96 kit, it is possible to run up to 96 samples simultaneously. Each sample's amplicons will be individually barcoded in the subsequent steps. It is strongly advised to refer to the current Oxford Nanopore Protocol for detailed instructions on these steps. As a tip, you can aliquot the Rapid barcodes into a PCR strip to facilitate multi-channeling. For comprehensive information, please consult the appropriate documentation.
 

SECTION: Rapid barcoding using the RBK110.96 kit for 96 samples
8. Add 7.5 µL of each diluted PCR reaction from step 15 to the labeled PCR tube.  
Set up the following reaction for each sample:


Component                                                                                                                                Volume
DNA amplicons from step 15 (100ng total)                                                        7.5 µL
Fragmentation Mix RB01-12 (one for each sample, included in kit)                  2.5 µL
Total                                                                                                                       10 µL

NB* Mix gently by flicking the tube, and spin down.
SECTION: Rapid barcoding using the RBK110.96 kit for 96 samples
9. Incubate the reaction in a thermocycler with the following settings:
 
20°C20 minutes65°C10 seconds4°C1 minute

Note: All PCR products now contain DNA barcodes that will be resolved during the sequencing process.
SECTION: Rapid barocoding using the SLK RBK109
9.1. Pool all barcoded samples, noting the total volume.
SECTION: Rapid barocoding using the SLK RBK109
9.2. Add an equal volume (1:1) of Ampure beads to the pooled sample tube and mix gently by either flicking or pipetting.

 

SECTION: MinION NGS sequencing
10. Prepare the flow-cells for sequencing. Prime the flow cell and add the priming fluid as recommend. 

Start the sequencing run using MinKNOW latest version. 

 https://community.nanoporetech.com/protocols/pcr-tiling-of-sars-cov-2-virus-with-rapid-barcoding-sqk-rbk110/v/PCTR_9125_v110_revA_24Mar2021. 
SECTION: Quantification of DNA using Qubit
1.1. Label the tube lids. Do not label the side of the tube as this could interfere with the sample reading. Use only thin-wall, clear, 0.5mL PCR tubes. Acceptable tubes include Qubit™ assay tubes (Cat. No. Q32856)