TITLE: Integrated Indexing One-Step RT-PCR with NEB LunaScript: Influenza A Virus Sequencing with Native Oxford Nanopore Software Compatibility
AUTHORS: Abhinaya Srikanth, Brian Merritt, Peter Thielen

[DESCRIPTION]
This integrated indexing RT-PCR protocol has been optimized for whole genome sequencing of influenza A virus (IAV) using Oxford Nanopore’s Ligation Sequencing Kit V14. The updated protocol is an optimization of an early approach used across several laboratories for research purposes, and uses New England Biolabs reagent sets including LunaScript‱ Multiplex One-Step RT-PCR (#E1555), NEBNext dA-Tailing Module (E6053), and the NEBNext Quick Ligation Module (E6056). Previously demonstrated IAV whole genome sequencing approaches for Oxford Nanopore used Superscript III/Platinum Taq RT-PCR reagents input into the Native Indexing reagent set, which results in a complex, multi-step process that requires more than a full working day to carry out. 
 
The improved integrated indexing protocol can be completed within a working day, and incorporation of the molecular index during first-strand cDNA synthesis in the RT-PCR step reduces the likelihood of sample cross-contamination during handling.  Recent improvements to the approach additionally include Oxford Nanopore defined constant regions that are required for full compatibility with default informatics packages. This update now enables native demultiplexing within either Guppy or Dorado, effectively serving as a “molecular patch” where previous implementations required software modifications that were often broken by silent updates to MinKNOW.   
 
As designed, this protocol has potential for extension to other viruses of interest by modifying primer sets. A simplified excel workbook is provided for design of new primer sets, for which the first worksheet requires input of gene-specific primer sets, the second worksheet provides fully constructed primers, and the third can be exported directly for submission to a primer synthesis vendor such as IDT DNA.

[BEFORE_START]
Note: High quality isolated Influenza A RNA must be used for this protocol as it is essential for successful complete genome amplification. There are many options for RNA purification, including solid phase reversible immobilization methods (e.g. spin columns, paramagnetic beads), and Trizol derivatives. Freshly isolated RNA with minimal or no freeze thaw cycles typically ensures the highest success rates for complete genome amplification.

[STEPS]
SECTION: Protocol for Integrated Indexing of Influenza A Virus
1. 
Prepare the One-Step Reverse Transcriptase Multi-Segment PCR for Influenza A Virus as described below:

 


SECTION: Protocol for Integrated Indexing of Influenza A Virus
2. Flick the tube or pipet up and down to mix at minimum 10 times followed by a short spin
SECTION: Protocol for Integrated Indexing of Influenza A Virus
3. Incubate reaction in thermo-cycler following these steps:

 



SECTION: 3.	Library Pooling and Purification
4. Quantify unpurified PCR product using Qubit High Sensitivity DNA reagents
SECTION: 3.	Library Pooling and Purification
5. Pool indexed PCR products together by equal mass into a single tube for purification


SECTION: 3.	Library Pooling and Purification
6. Add .6 volumes of AMPure XP to the pooled iiRT-PCR product and mix by finger flicking
 




SECTION: 3.	Library Pooling and Purification
7. Incubate for 5 min  at room temperature or if available incubate on hula mixer at low speeds or IKA plate shaker at 500 RPM

SECTION: 3.	Library Pooling and Purification
8. Move to magnetic stand to separate beads, allowing them to sit until solution clears
SECTION: 3.	Library Pooling and Purification
9. Remove and discard supernatant
SECTION: 3.	Library Pooling and Purification
10. Add 500 µL  of freshly made 80% ethanol

SECTION: 3.	Library Pooling and Purification
11. Remove tube from magnet and finger flick
SECTION: 3.	Library Pooling and Purification
12. Repeat steps 8-11 for a total of two washes
SECTION: 3.	Library Pooling and Purification
13. Allow to dry for 5 min  or until bead pellet is no longer shiny

 

SECTION: 3.	Library Pooling and Purification
14. Add 31 µL  nuclease-free water and resuspend with gentle pipetting. Incubate for 10 min .
 



SECTION: 3.	Library Pooling and Purification
15. Move to magnetic stand and allow to clear for 5 min 

SECTION: 3.	Library Pooling and Purification
16. Transfer purified DNA to new tube
SECTION: 3.	Library Pooling and Purification
17. Quantify 1 µL  of final pooled iiRT-PCR product with high sensitivity qubit reagents



SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
19. Thaw all NEB consumables on ice and once thawed allow mixture to adjust to room temperature. Then pipette the buffer up and down to. Break up the precipitate and then a 30 s  vortex. 

SECTION: 3.	Library Pooling and Purification
18. (Optional) Following purification of iiRT-PCR products, samples can be analyzed on an agarose gel, Tapestation, or similar platform.
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
20. Place 100-200 fmol of barcoded, pooled and purified DNA into a 1.5 Eppendorf tube and adjust volume to 30 µL  with nuclease free water


SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
21. Mix in a 0.2 ml PCR plate and then flick and spin down:


 


SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
22. Incubate plate in thermal cycler at 37 °C for 30 min and then 65 °C for   5 min 
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
23. Transfer DNA into 1.5 ml tubes
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
24. Add 60 µL of AMPure beads to the tube and flick to mix

SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
25. Incubate on hula mixer for 5 min 
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
26. Spin down and place on magnet. Once solution is clear pipette off supernatant
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
27. Add 200 µL of 70% ethanol and remove ethanol without disturbing beads

SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
28. Repeat step 27
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
29. Take tube off of magnet and spin down, then place back on magnet and remove any residual ethanol
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
30. Remove tubes from magnet and resuspend pellets in 61 µL  of nuclease free water

SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
31. Incubate for 2 min  at room temperature

SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
32. Place tubes on magnet and once solution is clear remove supernatant and place in clean 1.5 ml tubes
SECTION: DNA A-Tailing with NEBNext dA-Tailing Module
33. Quantify 1 µL  using qubit

SECTION: Adapter ligation and clean-up
34. Thaw all NEB reagents at room temperature, spin down for 5 seconds, and mix by pipetting. DON’T VORTEX T4 DNA Ligase. Spin down the Ligation Adapter and Quick T4 DNA Ligase, pipette to mix then place on ice. Thaw the Elution Buffer and Short Fragment Buffer at room temp, mix by
vortexing, spin down, and then place on ice.
SECTION: Adapter ligation and clean-up
35. Mix by pipetting and spin down the following reaction:




 




SECTION: Adapter ligation and clean-up
36. Incubate the reaction for 20 min  at room temperature

SECTION: Adapter ligation and clean-up
37. Transfer DNA into 1.5 ml tubes
SECTION: Adapter ligation and clean-up
38. Add 20 µL  of AMPure beads to each tube and flick to mix

SECTION: Adapter ligation and clean-up
39. Incubate on hula mixer for 5 min 

SECTION: Adapter ligation and clean-up
40. Spin down and place on magnet. Once solution is clear pipette off supernatant
SECTION: Adapter ligation and clean-up
41. Add 125 µL  of Short Fragment Buffer, flick to resuspend, then place back on tube and allow beads to pellet, then remove short fragment buffer without disturbing beads

SECTION: Adapter ligation and clean-up
42. Repeat step 41
SECTION: Adapter ligation and clean-up
43. Take tube off of magnet and spin down, then place back on magnet and remove any residual Buffer
SECTION: Adapter ligation and clean-up
44. Remove tubes from magnet and resuspend pellets in 15 µL of NFW

SECTION: Adapter ligation and clean-up
45. Incubate for 10 min  at 37 °C  and every 2 min  agitate by lightly flicking sample

SECTION: Adapter ligation and clean-up
46. Place tubes on magnet and once solution is clear remove supernatant and place in a clean 1.5 ml tube


 

SECTION: Final Library Quantification
47. Quantify 1 µL  of the final clean library using a Qubit DNA fluorometer

SECTION: Final Library Quantification
48. Optional: Run final library on a Tapestation to evaluate average peak size
SECTION: Final Library Quantification
49. Calculate the final volume required for 200fmol to be sequenced on a MinION flowcell
 

SECTION: Final Library Quantification
50. Proceed to load the Oxford Nanopore flowcell using the most up to date protocols from the vendor.