Dec 02, 2024
Optimized RT-PCR Protocols for Whole Genome Amplification of Influenza A Virus for NGS
- Iryna Goraichuk1,
- Jacquline Risalvato1,
- Mary Pantin-Jackwood1,
- David L. Suarez1
- 1USDA, ARS, US National Poultry Research Center
- NGS Optimization
Protocol Citation: Iryna Goraichuk, Jacquline Risalvato, Mary Pantin-Jackwood, David L. Suarez 2024. Optimized RT-PCR Protocols for Whole Genome Amplification of Influenza A Virus for NGS. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62r15gqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 21, 2024
Last Modified: December 02, 2024
Protocol Integer ID: 103796
Keywords: Influenza, WGS, Whole-genome sequencing, NGS, Next-generation sequencing, sequencing, Influenza A virus, whole genome, RT-PCR, one-step, Illumin, Nanopore, MinION, ONT, Oxford Nanopore Technologies, NEB, New England Biolabs, ThermoFisher, Thermo Fisher, Invitrogen, LunaScript, LunaScript Multiplex, LunaScript Multiplex One-Step RT-PCR Kit Protocol, SuperScript, SuperScript IV, SuperScript IV One-Step RT-PCR Systems, Zymo
Abstract
This protocol describes optimized protocols for simultaneous amplification of all 8 influenza A segments using the Opti primer set with the SuperScript IV one-step RT-PCR kit or LunaScript Multiplex One-Step RT-PCR Kit. Purified amplicons can be used for NGS library preparation for influenza whole-genome sequencing.
Materials & Consumables
Materials & Consumables
Materials
5 μl of Influenza A RNA
Consumables
- SuperScript IV One-Step RT-PCR System (Invitrogen, cat. number 12594100 or 12594025) or LunaScript Multiplex One-Step RT-PCR Kit (NEB, cat. number E1555S or E1555L)
- Opti primers:
| COMPONENT | SEQUENCE (5' -> 3') | |
| Opti F1 | GTT ACG CGC CAG CAA AAG CAG G | |
| Opti F2 | GTT ACG CGC CAG CGA AAG CAG G | |
| Opti R | GTT ACG CGC CAG TAG AAA CAA GG |
OPTI Primer Mix
OPTI Primer Mix
First, create a master 100 micromolar (µM) stock solution for each primer and then dilute it to a 20 micromolar (µM) working solution mix. This reduces the number of freeze/thaw cycles that the primer stock goes through and reduces the chances of contamination.
Preparation of Primer Stock Solutions (100 μM)
Note
The microliters of water required to create a 100 micromolar (µM) solution is 10x the nanomoles of lyophilized primer (i.e., if 26.5 nmol is noted on side of tube, a 100 μM primer stock solution is created by adding 265 µL of water or TE buffer to stock tube). The original primer tubes are used for a 100 µM stock.
- Find the oligo yield information in nmol on your tube label
- Multiply this number by 10. The resulting product is the amount of water or TE buffer needed (in µL)
- Reconstitute lyophilized primers in required volume of molecular grade water (i.e., RNase/DNase free) or TE buffer
- Once water has been added, vortex for at least 00:00:10 to ensure the lyophilized primers are fully dissolved and store at -20 °C to use as needed
10s
Preparation of Primer Working Solutions (20 μM)
In a clean template-free pre-PCR hood, prepare working solution of the OPTI primer mix:
- Allow stock solution to thaw completely at Room temperature
- Vortex the stock solution for at least 00:00:10 to ensure homogenization before adding it to the working solution
- Prepare a 20 micromolar (µM) working solution by combining stock solutions (100 µM) of Opti F1/F2/R primers in 0.35:0.65:1 ratio:
| COMPONENT | CONCENTRATION | VOLUME | |
| Nuclease-free water | - | 80 µl | |
| Opti F1 | 100 µM | 3.5 µl | |
| Opti F2 | 100 µM | 6.5 µl | |
| Opti R | 100 µM | 10 µl | |
| TOTAL | 100 µl |
Note
100 µl of Opti primer mix sufficient to perform 40 reactions at a 50-µL RT-PCR reaction volume
- Once the concentrated stock is added, vortex the new working solution for at least 00:00:10
- To maintain optimal use of the primers, limit freeze/thaw cycles, as this can lead to degradation over time. To avoid repeated thawing and freezing, prepare small aliquots of working solutions and store at -20 °C . Thawed working primer solutions can be kept at 4 °C for weeks or months to use as needed.
20s
Select one of the two one-step RT-PCR kit protocols below:
- SuperScript IV One-Step RT-PCR System (Invitrogen)
- LunaScript Multiplex One-Step RT-PCR Kit (NEB)
SuperScript IV One-Step RT-PCR System (Invitrogen)4 steps
Preheat the thermocycler to 55 °C , with the heated lid set to 105 °C
Keep all components, reaction mixes, and samples On ice
Prepare a 25 µL Master Mix (Volume 1) per reaction if visualization on TapeStation or no visualization is intended, and a 50 µL Master Mix (Volume 2) if gel visualization after PCR is planned for amplification product verification. Mix the following components:
| COMPONENT | VOLUME 1 | VOLUME 2 | |
| Nuclease-free water | 8.5 µl | 17 µl | |
| 2X Platinum™ SuperFi™ RT-PCR Master Mix | 12.5 µl | 25 µl | |
| Opti primer mix (20 µM working solution) | 1.25 µl | 2.5 µl | |
| SuperScript™ IV RT Mix | 0.25 µl | 0.5 µl | |
| RNA template | 2.5 µl | 5 µl | |
| TOTAL | 25 µl | 50 µl |
Mix gently, briefly centrifuge, and ensure all the components are at the bottom of the amplification tube
Place the reaction tubes in the pre-heated thermocycler and select the following RT-PCR cycling conditions:
| STEP | TEMPERATURE | TIME | CYCLE | |
| Reverse transcription | 55˚C | 2 min | 1 | |
| 42˚C | 90 min | |||
| RT inactivation/initial denaturation | 98˚C | 2 min | 1 | |
| Amplification 1 | 98˚C | 10 sec | 5 | |
| 45˚C | 30 sec | |||
| 72˚C | 3.5 min | |||
| Amplification2 | 98˚C | 10 sec | 30 | |
| 67˚C | 30 sec | |||
| 72˚C | 3.5 min | |||
| Final extension | 72˚C | 10 min | 1 | |
| Hold | 4˚C | ∞ | - |
Note
Safe Stop Point: If necessary, the protocol can be paused and samples can be kept at 4 °C overnight or at -20 °C for long-term storage, before proceeding to downstream applications.
For downstream NGS library construction, a cleanup step with Select-a-Size DNA Clean & Concentrator (Zymo Research, cat. number D4080) is recommended, following the manufacturer’s protocol to retain DNA fragments ≥ 300 bp.