TITLE: Hepatitis C Virus (HCV) subtype 1b sequencing protocol v.1
AUTHORS: Alí  F Ruiz-Higareda, Kame Galan, Julio  C Garza-Cabello, Ana G. Rivas-Estilla

[DESCRIPTION]
Amplicon sequencing protocol for Hepatitis C virus subtype 1b using Oxford Nanopore Technologies.

[STEPS]
SECTION: cDNA synthesis
1. Prepare the RNA-primer Mix following these instructions:
SECTION: cDNA synthesis
2. Prepare the RT reaction mix following the next steps:
3. Incubate the reaction as follows:
10 min   at 55 °C 
10 min  at 80 °C 

SECTION: cDNA synthesis
2.3. Combine and the RNA-primer and RT reaction mixes in a 0.2 mL tube, mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
SECTION: cDNA synthesis
1.1. Mix the following components in a 0.2 mL tube:
 
 

SECTION: cDNA synthesis
1.2. Mix and briefly centrifugate the components.
SECTION: cDNA synthesis
1.3. Heat the RNA-primer mix at 65 °C  for 5 min  and then incubate on ice  for at least 1 min 

SECTION: cDNA synthesis
2.1. Vortex and briefly centrifugate the 5X SSIV Buffer.
SECTION: cDNA synthesis
2.2. Combine the following components in a tube:

 


SECTION: cDNA synthesis
3. Mix and briefly centrifugate the contents.
2. 
SECTION: Genome amplification
4. The primer pools used in this section are described in "MATERIALS" Section.
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8 PCR tubes: 

 


SECTION: Genome amplification
5. Add 2.5 µL cDNA to each tube and mix well by pipetting.

SECTION: Genome amplification
6. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: Genome amplification
7. Set-up the following program on the thermal cycler:  

Step                       Temperature    Time                  Cycles
Heat Activation      94 °C       3 min  1
Denaturation          94 °C       30 s  35
Annealing               64 °C       45 s  35
Extension               72 °C       2 min  35
Final Extension      72 °C       10 min   1
Hold                       8 °C                     ∞             1

SECTION: Sequencing library preparation
9. DNA repair and end-prep.

Incubate for 15 min at 20 °C

1 µg of DNA* in 24 µL of nuclease free water
1.75 µL of NEB NextFFPE DNA Repair Buffer
1.75 µL of Ultra II End-prep reaction buffer
1 µL of NEB Next FFPE DNA Repair Mix
1 µL of NEB Next FFPE DNA Repair Mix
*DNA from previous step(PCR Products)
Inactivate the reaction for 15 minat 65 °C.

SECTION: Sequencing library preparation
10. Barcode ligation

Incubate for 20 min at 25 °C:
1.25 µLof Native Barcode
5 µL of Blunt/TA Ligase Master Mix
3.75 µL of end-prepped DNA

Inactivate the reaction for 10 min at 65 °C.

 

SECTION: Sequencing library preparation
11. Mix all the barcoded samples into a single 1.5 ml tube.

Purify products with AMPureXP beads:



SECTION: Sequencing library preparation
12. Adapter Ligation

Incubate for 20 min at Room temperature:

30 µL of pooled barcoded sample from previous step.
5 µL of Adapter Mix (AMII).
10 µL NEB NextQuick Ligation Reaction Buffer (X5).
5 µL Quick T4 DNA Ligase.



Incubate reaction for 20 min  at Room temperature .

SECTION: Genome amplification
8. PCR products can be verified using 1% agarose gels.
2. 
12. 
SECTION: Sequencing library preparation
11.1. Add resuspended AMPure XP beads to the pooled barcoded sample in a 1:1 relation, mix by flicking the tube.
SECTION: Sequencing library preparation
11.1. 
SECTION: Sequencing library preparation
11.2. Incubate on a Hula mixer for 5 min at Room temperature 

SECTION: Sequencing library preparation
11.3. Pellet on a magnetic rack until eluate is clear and colorless, with the cap open. Keep the tube on the magnet, and pipette off the supernatant.
 

SECTION: Sequencing library preparation
11.4. Wash the beads with 200 µL of SFB.

SECTION: Sequencing library preparation
11.5. Pellet on a magnetic rack until eluate is clear and colorless, with the cap open. Keep the tube on the magnet, and pipette off the supernatant.
 

SECTION: Sequencing library preparation
11.6. Resuspend the pellet with 30 µL of nuclease free water and incubate it for 2 min at Room temperature 

SECTION: Sequencing library preparation
11.7. Pellet on a magnetic rack until eluate is clear and colorless, for at least 1 min.

SECTION: Sequencing library preparation
11.8. Remove and retain 30 µL of the elute in a 1.5 mL tube.

SECTION: Sequencing library preparation
13. Purify product with AMPureXP beads:



SECTION: Sequencing library preparation
13.1. Add 50 µL of resuspended AMPure XP beads to the reaction, mix by flicking the tube.

SECTION: Sequencing library preparation
13.2. Incubate on a Hula mixer for 5 min at Room temperature 

SECTION: Sequencing library preparation
13.3. Pellet on a magnetic rack until eluate is clear and colorless, with the cap open. Keep the tube on the magnet, and pipette off the supernatant.
 

SECTION: Sequencing library preparation
13.4. Wash the beads with 250 µL of SFB.

SECTION: Sequencing library preparation
13.5. Pellet on a magnetic rack until eluate is clear and colorless, with the cap open. Keep the tube on the magnet, and pipette off the supernatant.
 

SECTION: Sequencing library preparation
13.6. Resuspend the pellet with 13 µL of elution buffer(ONT) and incubate it for 10 min at Room temperature 

SECTION: Sequencing library preparation
13.7. Pellet on a magnetic rack until eluate is clear and colorless, for at least 1 min.

SECTION: Sequencing library preparation
13.8. Remove and retain 13 µL of the elute in a 1.5 mL tube.

SECTION: Priming and loading the SpotON flow cell.
14. 
 

Add 30 μl of Flush Tether (FLT) directly to the tube of the Flush Buffer (FB), and pulse spin the tube to collect liquid at the bottom of the tube.

SECTION: Priming and loading the SpotON flow cell.
15. Open the MinION device lid and slide the flow cell under the clip.
SECTION: Priming and loading the SpotON flow cell.
16. Slide the priming port cover to open the priming port.
SECTION: Priming and loading the SpotON flow cell.
17. Check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few µL):
SECTION: Priming and loading the SpotON flow cell.
17.1. Set a P1000 pipette to 200 μl.
SECTION: Priming and loading the SpotON flow cell.
17.2. Insert the tip into the priming port.
SECTION: Priming and loading the SpotON flow cell.
17.3. Turn the wheel  220-230 µL, to draw back 20-30 µL, or until you can see a small volume of buffer entering the pipette tip.
SECTION: Priming and loading the SpotON flow cell.
18. Load 800 μl of the priming mix into the flow cell via the priming port.

 

SECTION: Priming and loading the SpotON flow cell.
19. Wait for 5 minutes.
SECTION: Priming and loading the SpotON flow cell.
20. While waiting prepare the library as follows: 
   ComponentVolume1Sequencing Buffer (SQB)37.5 µL2Loading Beads (LB), mixed immediately before use25.5 µL3DNA library12 µL


SECTION: Priming and loading the SpotON flow cell.
21. Gently lift the SpotON sample port cover.
SECTION: Priming and loading the SpotON flow cell.
22. Load 200 μl of the priming mix into the flow cell via the priming port.

 

SECTION: Priming and loading the SpotON flow cell.
23. Mix the prepared library gently by pipetting up and down.
SECTION: Priming and loading the SpotON flow cell.
24. Add 75 µL of sample to the flow cell via the SpotON port in a dropwise fashion. 
 


SECTION: Priming and loading the SpotON flow cell.
25. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.
SECTION: MinION Software
26. While starting the sequencing run using MinKNOW, the following parameters are to be selected:
SECTION: MinION Software
26.1. Kit: SQK-LSK109.

Barcode kit: EXP-NBD196, turn on Live basecalling, ensure to turn on double-ended barcoding in the basecalling settings

Run length: Set the run length to a minimum of 12 hours (you can stop the run once sufficient data has been collected or you can select more time if deemed necessary). 

Basecalling: On and select 'fast basecalling'. High accuracy can be selected, however it would require to basecall more time since it is more demanding task for the computer.