TITLE: HEP-TILE: HBV whole genome sequencing (nanopore protocol)
AUTHORS: Sheila Lumley, Chris Kent, Josh Quick, Philippa Matthews

[DESCRIPTION]
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 are here: https://github.com/quick-lab/primerschemes/blob/main/primerschemes/hbv/600/v2.1.0/primer.bed

The amplicons can also be fragmented and sequenced on Illumina platforms.


[BEFORE_START]
Prepare between 11 and 95 DNA samples plus 1 negative control using this protocol.

[STEPS]
SECTION: Multiplex PCR
5. 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.



 



 

SECTION: Multiplex PCR
6. Add 2.5 µL cDNA to each of the PCR reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
 

 

SECTION: Multiplex PCR
7. Set-up the following program on the thermal cycler:  

Step                        Temperature     Time                    Cycles

Heat Activation     98 °C        30 s    1
Denaturation         98 °C        15 s     35 
Annealing              65 °C        5 min     35
Hold                       4 °C            Indefinite              1




SECTION: Bead clean up
8. Label strip-tubes/plate and combine the following volumes of each PCR reaction for 25 µL each sample:

 



 

SECTION: End preparation
10. In a new PCR strip-tube/plate set up the following reaction for each sample, normalising to 80ng amplicon DNA input per sample:


SECTION: Native barcoding
12. Quantify 1 µLof 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.



14. In a new 1.5ml eppendorf or PCR tube set up the following adapter ligation reaction


 




SECTION: MinION sequencing
20. Thoroughly mix the contents of the library beads by pipetting, it is vial that they are mixed immediately before use. 


 

SECTION: Primer pool preparation
2. 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.

SECTION: Primer pool preparation
4. Dilute 100 micromolar (µM)  pools 1:10 in molecular grade water, to generate 10 micromolar (µM) primer stocks.

 

 

SECTION: Primer pool preparation
3. Sort all odd regions primers into one or more tube racks. Add 5 µL of each odd region primer to a 1.5 mLEppendorf 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.

 

 

SECTION: Sample preparation
1. Prepare between 11 and 95 DNA samples plus 1 negative control of nuclease-free water per library. If previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on ice at all times.


 

SECTION: Bead clean up
9. Add 40 µLSPRI beads to 50 µLpooled PCR product for an 0.8x clean up. 
Gently mix and incubate for 5 min at room temperature

SECTION: Bead clean up
9.1. Place on a magnetic rack and incubate for 2 minor until the beads have pelletted and supernatant is completely clear. Remove and discard the supernatant.

SECTION: Bead clean up
9.2. Perform an ethanol wash: add 100 µLof room temperature 70% ethanol to bathe the pellet, carefully remove and discard ethanol being careful not to touch the bead pellet.

SECTION: Bead clean up
9.3. Repeat the ethanol wash (step 9.2)
SECTION: Bead clean up
9.4. Pulse centrifuge to collect and remove residual ethanol, remove excess with a P10 pipette, it is very important all is removed.
SECTION: Bead clean up
9.5. With the tube lid open, air dry for 1-2 minutes, or until the pellet loses its shine. Do not over-dry.
SECTION: Bead clean up
9.6. Re-suspend pellet in 20 µLH20, mix gently and incubate for 2 minutes at room temperature

SECTION: Bead clean up
9.7. Place on magetic rack, transfer the supernatant to clean labelled tubes
SECTION: Bead clean up
9.8. Quantify using a Qubit or Quantus.
SECTION: End preparation
10.1. 
 

 



SECTION: End preparation
10.2. Incubate at room temperature for 15 min
Incubate at 65 °Cfor 15 min
Incubate on ice for 1 min

SECTION: Native barcoding
11. In a new PCR strip-tube/plate set up the following reaction for each sample:
SECTION: Native barcoding
11.1. 
 

 

SECTION: Native barcoding
11.2. Incubate at room temperature for 20 min


SECTION: Native barcoding
11.3. Add EDTA and mix thoroughly


 

 

 

SECTION: Native barcoding
11.4. In a new 1.5ml Eppendorf tube pull all one-pot barcoding reactions together.

 

SECTION: Native barcoding
11.6. Add ampure beads (supplied with ONT kit) for a 1.5x clean up. If very large volume split between 2 tubes

Example:


 


 


SECTION: Native barcoding
11.5. Determine volume of pooled barcoded samples, add an equal volume of water
SECTION: Native barcoding
11.7. Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 5 min  at room temperature.

SECTION: Native barcoding
11.8. Place on magnetic rack and incubate for 2 min   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.

SECTION: Native barcoding
11.9. 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.
SECTION: Native barcoding
11.10. Repeat step 11.9 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
 

SECTION: Native barcoding
11.11. 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.
 


SECTION: Native barcoding
11.12. 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.
SECTION: Native barcoding
11.13. With the tube lid open incubate for 1 min  or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

SECTION: Native barcoding
11.14. 2 min Re-suspend pellet in 31 µLnuclease free water, mix gently by either flicking or pipetting and incubate for 2 min

SECTION: Native barcoding
11.15. Place on magnet and transfer sample to a clean 1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.
SECTION: Adaptor ligation
13. Set up the following Native Adapter (NA) ligation and clean-up with SFB.
14.1. Incubate at room temperature for 20 min

15. Perform a bead clean up. 
15.1. 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 5 min  at room temperature.

15.2. Place on magnetic rack and incubate for 2 min   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.

15.3. 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.
15.4. Repeat step 15.3 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
 

15.5. Re-suspend pellet in 15 µLEB (ONT), mix gently by either flicking or pipetting and incubate for 2 min

15.6. Place on magnet and transfer sample to a clean 1.5 mL  Eppendorf tube ensuring no beads are transferred into this tube.
SECTION: Flow cell check and priming
17. Refer to ONT documentation for images explaining how to check, prime and load flow cell.
SECTION: Flow cell check and priming
18. 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.
SECTION: Flow cell check and priming
19. 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:

 

SECTION: Flow cell check and priming
19.1. Slide the flow cell priming port cover clockwise to open the priming port
SECTION: Flow cell check and priming
19.2. 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. 
SECTION: Flow cell check and priming
19.3. Visually check that there is continuous buffer from the priming port across the sensor array.
SECTION: Flow cell check and priming
19.4. Load 800 µLof 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.

SECTION: MinION sequencing
21. 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):


 

SECTION: MinION sequencing
22. 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

SECTION: MinION sequencing
23. Mix the prepared library gently by pipetting just prior to loading
SECTION: MinION sequencing
24. 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.
SECTION: MinION sequencing
25. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
SECTION: MinION sequencing
26. Place the light shield onto the flow cell. And close the device lid.
16. Quantify the final library using a fluorometer such as a Qubit or Quatus. 
 



 


SECTION: MinION sequencing
27. Start the sequencing run using MinKnow.