TITLE: Whole-Genome Amplification of Respiratory Syncytial Virus (RSV) using Illumina CovidSeq reagents for Next-Generation Sequencing
AUTHORS: Carlos Davina-Nunez, Sonia Perez-Castro, Montse Godoy-Diz, Benito Regueiro-Garcia

[DESCRIPTION]
Protocol for amplification of RSV and for whole-genome sequencing. This protocol can amplify the whole genome in two parallel RT-PCR reactions in order to make RSV sequencing accessible and affordable. This protocol does not require prior subtyping as it covers RSV-A and RSV-B in the same reaction. The primer panel is an optimisation of a previously published panel by Wang et al. 

Our optimisation from the original panel allows for multiplex PCR, so the whole genome can be amplified in just two PCR reactions. In addition to this, primers have been modified to account for commonly-occurring mutations that affect primer-binding areas and were causing suboptimal amplification. These primers were used to cover the complete hRSV genome (both A and B) by splitting them into two pools of non-consecutive amplicons (odd-numbered amplicon primers in one pool, even-numbered amplicon primers in other).

This protocol has been tested for RSV RNA amplification from RSV-positive nasopharyngeal swabs of CT value up to 26 using Seegene Allplex Respiratory Panel (Seegene Inc, Seoul, South Korea). However, it provides best results with CT values below 21. For samples of low viral load, an alternative primer panel is provided only with the amplicons sufficient to sequence the G and F genes. This alternative panel has succesfully sequenced samples of CT values up to 30, albeit with higher amount of total reads required in the case of very high CT value.

Illumina CovidSeq (Illumina Inc, San Diego, USA) reagents were used for the RT-PCR, with a mix previously published for amplification of Influenza RNA and a thermocycling program optimised in our lab. Library preparation was performed according to the Illumina CovidSeq protocol.

v3 (2024.02.22)

Modifications from v2: 
1. Primer A10f has been replaced to solve the amplicon dropout for Nextclade variant A.D.5.2 (mutation G13793A).
2. The relative concentration of primers has been modified to improve amplicon homogeneity.
3. An alternative panel has been added to amplify the G and F genes suitable for low viral load samples.
4. A bioinformatic analysis section has been added with a link to a custom pipeline for this protocol.


[BEFORE_START]
This protocol was tested using as input RNA extracted from nasopharyngeal swabs after confirmation of RSV infection via RT-PCR. Samples were extracted using the QIASymphony DSP Virus/Pathogen Midi Kit (Qiagen, Hilden, Germany).

[STEPS]
SECTION: Primer pools preparation
1. Pools WG: for samples of high viral load, covering the whole RSV genome.

For a final concentration of 10uM: add 607 ul of Nuclease-Free water to Pool 1 and 639 ul to Pool 2.
 
 
 

Pools GF: for samples of mid-to-low viral load, covering the G and F genes.

For a final concentration of 10uM: add 256 ul of Nuclease-Free water to Pool 1 and 270 ul to Pool 2.

 
 

SECTION: RT-PCR
2. Two Master Mixes must be prepared per sample: one for Pool1 and one for Pool 2 (Table 2). Manipulate reagents according to the Illumina CovidSeq Reference Guide. The protocol is the same for primers WG or primers GF.

 

In a PCR tube, mix 20 ul of MasterMix with 5 ul of extracted RNA.

Place all tubes (two per sample) in a thermocycler and run the following program (Table 3):

 

SECTION: (OPTIONAL) Check RT-PCR result with Agilent Bioanalyzer
3. Use an Agilent Bioanalyzer to check for amplification peaks. Expect PCR peaks around ~2000 bps (Figure 1).


 



SECTION: Library preparation
4. Mix 10ul of tube one and tube two on each sample for a final 20 ul of PCR product. Follow instructions of the Illumina CovidSeq Reference Guide to generate sequencing-ready libraries.

Recommended: To ensure optimal normalisation, perform the library Clean-up on each tube and normalise individually instead of pooling. This improves normalisation especially in the presence of low-concentration PCR products.

Quantify samples after Clean-up using Qubit Flex and normalise samples.

(OPTIONAL): Check library preparation on an Agilent Bioanalyzer. The pattern expected is the usual post-tagmentation pattern from Illumina libraries with the highest peak around ~330bps.
SECTION: References
7. The illumina CovidSeq protocol can be found in:

Illumina CovidSeq Reference Guide

The primers found in Table 1 were obtained from:

 

 

The Master Mix used for RT-PCR with Illumina CovidSeq was first published in:

 

SECTION: Expected results
5. The following results were obtained after sequencing the samples on an Illumina NextSeq 1000 (Table 5).

Ct value for Whole-Genome amplification ranged from 15-21, while for G and F amplification was from 20 to 27. Average percentage of Illumina reads mapped to RSV was 35,52% for whole genome and 12,73% for GF amplification.

Average number of total reads was 195k for whole genome and 56k for GF, meaning that 41 and 143 samples could be sequenced in a single iSeq 100 run respectively (expecting 8M reads per run, as specified by the manufacturer).


 





SECTION: Bioinformatic Analysis
6. A pipeline for RSV sequencing analysis is provided in https://github.com/OMIC-G/RSV.

This pipeline uses Illumina reads as input and includes the BED file with the primers in this version for analysis, as well as the reference genomes recommended for the current season. The pipeline does not require prior categorization in RSV-A and B as it sorts reads in RSV-A and RSV-B.

Output provided from the pipeline includes:
1. RSV typing in RSV-A and RSV-B.
2. Two fasta files with all RSV-A and RSV-B consensus sequences.
3. A report including quality data and clade designation.
4. CLI-based upload to epiRSV GISAID database.