TITLE: NEBNext iiMS Influenza A DNA Library Prep for Oxford Nanopore
AUTHORS: Kaylinnette Pinet, Abhinaya Srikanth, Peter Thielen, New England   Biolabs, Juliet Bonnevie

[DESCRIPTION]
The NEBNext iiMS Influenza A module for Library Prep (Oxford Nanopore Technologies) includes universal Influenza A primer pairs with indexing tails that allow for generation of indexed full length Influenza A genome segments. The NEBNext iiMS Influenza A Library Prep (ONT) protocol incorporates Oxford Nanopore Native Barcoding sequence during targeted cDNA synthesis and amplification, allowing for the pooling of up to 48 samples directly after this initial reaction. The samples can be demultiplexed using the standard Oxford Nanopore Technologies settings.
 
Where larger volumes, customized or bulk packaging are required, we encourage consultation with the Customized Solutions team at NEB. Please complete the NEB Custom Contact Form at www.neb.com/CustomContactForm to learn more.
 
Figure 1. Workflow for NEBNext iiMS Influenza A Library Prep for ONT library preparation.
 



[BEFORE_START]
Please review the important information under the "Guidelines & Warnings" tab before beginning. 

[GUIDELINES]
Safe Stop Point: This is a point where you can safely stop the protocol. 

Caution: This signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

(Color): The color in parentheses indicates the cap color of the reagent to be added to a reaction. 

[STEPS]
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
1. Note:
1) We recommend setting up the RT-PCR reactions in a room (and ideally in a hood) separate from the library construction area to minimize cross-contamination of future reactions. 
2) We recommend inputs of ≥ 1,000 copies of the Influenza A viral genome.  
3) The presence of carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful. We recommend that the NTC reactions are prepared prior to non-NTC samples following the guidelines outlined below. 
4) Precipitates may appear in the LunaScript Multiplex One-Step RT-PCR Reaction Mix upon thawing, resuspend completely prior to use by vortexing or pipetting up and down 10 times. 
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
2. Briefly centrifuge the LunaScript Multiplex One-Step RT-PCR Enzyme Mix to collect solution to bottom of the tube, then place on ice. 
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
3. Thaw the LunaScript Multiplex One-Step RT-PCR Reaction Mix, NEBNext iiMS Flu A Primer Pairs 1-48, and the Nuclease-free Water at room temperature, then place on ice. Prior to use, vortex and briefly centrifuge the Lunascipt Multiplex One-Step Reaction Mix and the NEBNext iiMS Flu A Primer Pairs.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
4. For no template controls, mix the following components:
 
Note: Cover/ seal the NTC after mixing. 
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
5. Prepare individual cDNA synthesis and amplification reaction as described below:
For Pool Set A: 
 
* NEB recommends ≥ 1,000 copies of Influenza A viral genomes as input for high genome coverage.
**A master mix of the LunaScript Multiplex One-Step RT-PCR Reaction Mix, Lunascript Multiplex One-Step RT-PCR Enzyme Mix, and Nuclease-free Water can be prepared and stored at 4°C for up to 8 hours, or at -20°C for 24 hours prior to use.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
6. Mix reactions gently by pipetting up and down or by inverting the tubes, then briefly centrifuge to collect solutions to the bottoms of tubes.
Note: We recommend proceeding with the cDNA amplification and library construction in a different area or room.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
7. Place the tube in a thermal cycler with the heated lid set to 105°C or on and perform PCR amplification using the following PCR cycling conditions:
 
* A 55°C reverse transcription step temperature is optimal for the WarmStart Reverse Transcriptase. To ensure best performance and full WarmStart Reverse Transcriptase activation, avoid using a reverse transcription temperature below 50°C.
** The annealing temperature is primer-mix dependent, 45°C is optimal for the Influenza A homologous regions of the NEBNext iiMS Flu A Primers and 65°C is optimal for the full-length NEBNext iiMS Flu A Primers.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
8. Make a cDNA pool following Option A (Step 9) for an equi-volume pool or Option B (Step 10) for an equi-molar pool.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
9. Option A:  Make an equal volume cDNA pool by adding 10 - 25 µl of each of the indexed amplified cDNA products to a 1.5 ml DNA LoBind tube. Quantify the pool with a TapeStation or Qubit using a 1/10 dilution (1 µl cDNA pool: 9 µl Nuclease-free Water) of the cDNA pool.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
10. Option B: Quantify yields for each indexed amplified samples with a TapeStation or Qubit using 1/10 dilutions (1 µl cDNA: 9 µl Nuclease-free Water) of each cDNA sample. Make an equal molar cDNA pool by adding up to 20 µM of each of the indexed amplified cDNA products into a 1.5 ml DNA LoBind Tube.
SECTION: Protocol for use with Lunascript Multiplex One-Step RT-PCR Kit (E1555 S/L) with NEBNext iiMS Influenza A Primer Pairs 1-48
11. Safe Stop Point: Samples can be stored at 4°C overnight or –20°C for long term storage if they are not used immediately.
SECTION: Cleanup of pooled indexed cDNA amplicons
12. Notes: 
1) Use the pooled indexed cDNA samples from Step 9 or 10; a pool volume of 120 - 480 µl is recommended for bead cleanup. Any remaining cDNA can be stored long-term at -20°C.
2) The volumes of AMPure XP Beads described here are for use with the sample contained in the exact buffer at this step. Allow the beads to warm to room temperature for at least 30 minutes before use. These volumes may not work properly for a cleanup at a different step in the workflow. SPRIselect beads may also be used.

SECTION: Cleanup of pooled indexed cDNA amplicons
13. Vortex AMPure XP Beads to resuspend.
SECTION: Cleanup of pooled indexed cDNA amplicons
14. Add 0.6X resuspended beads to pooled barcoded samples from Step 8 (For example, if total pool volume is 480 µl, then add 288 µl of resuspended AMPure XP Beads). Mix well by flicking the tube or pipetting up and down 10 times, do not vortex. If necessary, perform a quick spin for 1 second to collect all liquid from the sides of the tube.
SECTION: Cleanup of pooled indexed cDNA amplicons
15. Incubate samples on bench top for 5 min at room temperature.

SECTION: Cleanup of pooled indexed cDNA amplicons
16. Place the tube on a 1.5 ml magnetic stand (such as NEB #S1506) to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube before placing on the magnetic stand.
SECTION: Cleanup of pooled indexed cDNA amplicons
17. After 3 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
SECTION: Cleanup of pooled indexed cDNA amplicons
18. Wash the beads by adding 200 to 500 μl of 80% freshly prepared ethanol to the tube while on the magnetic stand. Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. 
SECTION: Cleanup of pooled indexed cDNA amplicons
20. Perform a quick spin and place the sample tube on the magnetic stand, remove any residual ethanol.
SECTION: Cleanup of pooled indexed cDNA amplicons
21. Air dry the beads for 30 seconds while the tube is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

 

SECTION: Cleanup of pooled indexed cDNA amplicons
22. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of Nuclease-free Water
SECTION: Cleanup of pooled indexed cDNA amplicons
23. Resuspend the pellet by flicking the tube or pipetting up and down 10 times to mix. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample for 1 second to collect the liquid from the sides of the tube before placing back on the magnetic stand.

SECTION: Cleanup of pooled indexed cDNA amplicons
24. Place the tube on the magnetic stand. After 2 minutes (or when the solution is clear), transfer 32 μl to a PCR tube.
Assess the concentration of the purified barcoded DNA sample. We recommend using a Qubit fluorometer for accurate concentration assessment and TapeStation for cDNA size confirmation. Nanodrop is NOT recommended since it may overestimate the DNA concentration. Use 1 µl of the eluant to make a 1/10 dilution (1 µl eluant : 9 µl Nuclease-free Water) of the pool for the Qubit fluorometer and TapeStation analyses.
Safe Stop Point: Samples can be stored long term at –20°C if they are not used immediately.
SECTION: Cleanup of pooled indexed cDNA amplicons
25. Note: 
The cleaned-up pooled cDNA amplicons may be run on a TapeStation to confirm 900 – 2,500 bp amplicon sizes. To run on a TapeStation, dilute 1 µl of the cleaned-up pooled amplicons 10-fold with Nuclease-free Water and run 2 µl on a High Sensitivity D5000 DNA ScreenTape. (See Figure 1.1 below for example of amplicon size profile).

Figure 1.1: Examples of amplicons prepared from pooled samples for 10,000 genome copies of Influenza A or non-template control.


 

SECTION: NEBNext A-tailing
26. Add the following components to a sterile nuclease-free tube on ice:
 
*Note: NEB recommends cDNA input of 50 - 150 ng for this A-tailing reaction.
SECTION: NEBNext A-tailing
27. Flick the tube or pipette up and down 10 times to mix the solution. Perform a quick spin to collect all liquid from the sides of the tube. A small number of bubbles in the reaction will not inhibit performance.
SECTION: NEBNext A-tailing
28. Place in a thermal cycler, with the heated lid set to ≥ 75°C or on, and run the following program:
30 min at 37°C
5 min at 65°C 
Hold at 4°C
Proceed Immediately to Adapter Ligation in Step 29.


SECTION: Adaptor Ligation
29. Add the following components directly to A-tailed cDNA:
 
* Mix the NEBNext Quick Ligation Reaction Buffer by pipetting up and down several times prior to adding to the reaction.
** Ligation Adapter is provided in Oxford Nanopore Technologies Ligation Sequencing Kit(s); Ligation Sequencing Kit V14 (SQK-LSK114) or Ligation Sequencing Kit XL V14 (SQK-LSK114-XL).
SECTION: Adaptor Ligation
30.      
Flick the tube to mix solution. Perform a quick spin for 1 second to collect all liquid from the sides of the tube. 
(Caution: The NEBNext Quick Ligation Reaction Buffer is viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).
SECTION: Adaptor Ligation
31. Incubate at 25°C or room temperature for 20 minutes and proceed to cleanup of Adapter-ligated DNA in Step 32.
SECTION: Cleanup of Adapter Ligated DNA
32. Note: The volumes of AMPure XP Beads described here are for use with the sample contained in the exact buffer at this step. Allow the beads to warm to room temperature for at least 30 minutes before use. These volumes may not work properly for a cleanup at a different step in the workflow. SPRIselect beads may also be used
SECTION: Cleanup of Adapter Ligated DNA
33.      
Vortex AMPure XP Beads to resuspend.
SECTION: Cleanup of Adapter Ligated DNA
34.      
Add 27.5 μl (0.5X) of resuspended beads to the ligation mix. Mix well by flicking the tube followed by a quick spin for 1 second.
SECTION: Cleanup of Adapter Ligated DNA
35. Incubate samples for 5 min at room temperature.

SECTION: Cleanup of Adapter Ligated DNA
36. Place the tube on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Cleanup of Adapter Ligated DNA
37.      
After 3 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).
SECTION: Cleanup of Adapter Ligated DNA
38. Wash the beads by adding 125 μl of Short Fragment Buffer (SFB) provided in the Ligation Barcoding Kit from Oxford Nanopore Technologies. Flick the tube to resuspend pellet. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand. Place the tube on an appropriate magnetic stand.
SECTION: Cleanup of Adapter Ligated DNA
39. Wait for 3 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
SECTION: Cleanup of Adapter Ligated DNA
40. Repeat steps 38 and 39 once for a total of 2 washes. Remove all visible liquid after the second wash. If necessary, briefly spin the tube/ plate, place back on the magnet and remove traces of SFB with a p10 pipette tip. Once the SFB has been removed, proceed to Step 41.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. If beads have turned light brown and start to crack, they are too dry. 
SECTION: Cleanup of Adapter Ligated DNA
41. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 15 μl of Elution Buffer (EB) provided in the Ligation Barcoding Kit from Oxford Nanopore Technologies. 
SECTION: Cleanup of Adapter Ligated DNA
42. Resuspend the pellet well in EB buffer by flicking. Incubate for 5 min at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Cleanup of Adapter Ligated DNA
43. Place the tube/ plate on the magnetic stand. After 3 minutes (or when the solution is clear), transfer 15 μl to a new DNA LoBind tube.
SECTION: Cleanup of Adapter Ligated DNA
44. Use Qubit to quantify 1 μl of DNA sample. Follow Oxford Nanopore Technologies Ligation Sequencing Kit Protocol for preparing the flow cell and DNA library sequencing mix using 30 ng - 60 ng adapter-ligated cDNA sample (Step 43).
Note: After normalizing the DNA to 30 ng - 60 ng, if the volume is less than 12 µl, then bring up the sample volume to 12 µl with EB.

SECTION: Cleanup of pooled indexed cDNA amplicons
19. Repeat the wash once to make it a total of 2 washes.