Complete Hepatitis B Virus Sequencing using an ONT-Based Next-Generation Sequencing Protocol

Prepare the two standards calibrate the Qubit Fluorometer using Qubit dsDNA HS Assay kit Thermo Fisher Scientific (Qubit® dsDNA HS Reagent).

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)

For standards [STD]: Aliquot 190µL of Qubit® dsDNA HS Reagent working solution to each 500 µL thin-walled polypropylene tubes .

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

Add 10µL of STD 1 to the aliquoted 190µL.

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

Incubate at room temperature for0h 2m 0s prior to measuring the concentation using Qubit Fluorometer. **

After calibrating the machine with STD 1 & 2 proceed to measuring DNA for samples.

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

Add1µLof the specific sample to each well containing the pre-allocated 190µL of sample.

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

Incubate at room temperature for 0h 2m 0s. Measure the concentation using Qubit Fluorometer.

After calibrating the machine proceed to measuring DNA for samples.

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

Add1µLof the specific sample to each well containing the pre-allocated 190µL of sample.

Pulse vortex for 0h 2m 0sseconds to mix thoroughly without producing bubbles.

Incubate at room temperature for 0h 2m 0s. 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) 0h 2m 0s; the Qubit® dsDNA HS Reagent are light sensitive.**

DNA quantification using the Qubit fluorometer

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.

A	B	C
Primer Name	Sequence	Positions
SC_1_LEFT	TTC CAC CAA GCT CTG CAA GATC	11 - 32
SC_1_RIGHT	AGAGGAATATGATAAAACGCCGCA	384-407
SC_2_LEFT	CATCATCATCAT CACCA CCTCC	325-346
SC_2_RIGHT	AAAGCCCTACGAACCACTGAAC	692-713
SC_3_LEFT	AAATACCTATGGGAGTGGGCCT	632-653
SC_3_RIGHT	TTGTGTAAATGGAGCGGCAAAG	1 655-1 676
SC_4_LEFT	AGAAAACTTCCTGTTAACAGACCTATTG	949-976
SC_4_RIGHT	GGACGACAGAATTATCAGTCCCG	1 326-1 348
SC_5_LEFT	TCCATACTGCGGAACTCCTAGC	1 265-1 286
SC_5_RIGHT	TGTAAGACCTTGGGCAGGATTTG	1 632-1 654
SC_6_LEFT	CTTCTCATCTGCCGGTCCGTGT	1 559-1580
SC_6_RIGHT	AGA AGT CAG AAG GCA AAC GAGA	1 947-1 970
SC_7_LEFT	GGCTTTGGGGCATGGACATT	1 890-1 909
SC_7_RIGHT	ATCCACACTCCGAAAGAGACCA	2 256-2 277
SC_8_LEFT	GACAACTATTGTGGTTTCATATTTCT	2 193-2 218
SC_8_RIGHT	TTGTTGACACCTATTAATAATGTCCTCA	2 576-2 594
SC_9_LEFT	TGGGCTTTATTCCTCTACTGTCCC	2 492-2 515
SC_9_RIGHT	GGGAACAGAAAGATTCGTCCCC	2 889-2 910
SC_10_LEFT	TTGCGGGTCACCATATTCTTGG	2 816-2 837
SC_10_RIGHT	GGCCTGAGGATGACTGTCTCTT	3 189-3 210

Table1: Primers for Pool 1 & 2. Pool One are odd Numbers (SC_1, SC_3, ...) and Pool two are even Numbers (SC_2, SC_4,..)

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 for0h 15m 0s.

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.

1. To create a 100 µM stock of the primer pool for Pool 1, combine 5 µL of Pool 1 with 485µL of nuclease-free water in a labeled 1.5 mL Eppendorf tube called "Pool 1 (100 μM)". This will yield a total volume of 490µL, 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.

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.

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 for0h 15m 0s .

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.

A	B	C
Component	PCR 1	PCR 2
Q5 2x Master Mix	12.5 μL	12.5 μL
Primer pool 1 (10µM)	1.1 μL	-
Primer pool 2 (10µM)	-	1.1 µL
Nuclease-free water	8.9 μL	8.9 µL
DNA template	2.5 µL	2.5 µL
Total Volume	25µL	25µL

Table 2. PCR mastermix

In clean MasterMix cabinet:

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

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

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

4. Aliquot the 22.5µL of master-mix in labelled PCR strip tubes and transfer the master-mixes to the decontaminated #* extraction hood.

In the extraction and sample addition cabinet:

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

• Add 2.5µL each DNA sample to a tube containing 22.5µL Pool1.

• Add 2.5µL each DNA sample to a tube containing 22.5µL 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.

Incubate both PCR reactions in a thermocycler with the following settings:

A	B	C
Heat Activation	98°C	30 seconds
Denaturation	98°C	15 seconds
Annealing	65°C	5 minutes
Repeat denaturation and annealing for a total of 25 cycles
Hold	4°C	∞

Tabe 3: Tiling PCR conditions

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 20µL

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

"To add the lab chip preparation protocol"

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.

Add 7.5µL 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 10µL

NB* Mix gently by flicking the tube, and spin down.

Incubate the reaction in a thermocycler with the following settings:

A	B
20°C	20 minutes
65°C	10 seconds
4°C	1 minute

TABLE 4:

Note : All PCR products now contain DNA barcodes that will be resolved during the sequencing process.

Pool all barcoded samples, noting the total volume.

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.

Resuspend pellet in 30 µLElution Buffer (EB), mix gently by either flicking or pipetting and incubate for 00:02:00.

Place on magnetic stand and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.

Measure the concentration of samples using Qubit. ( See Section 1 ).

Add an equal volume (1:1) of Ampure beads to the pooled sample tube and mix gently by either flicking or pipetting.

Amplicon clean-up using SPRI beads for RAPID nanopore kit RBK004

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 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 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.

Repeat Step above.

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.

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.