Feb 22, 2024

Public workspaceWhole-Genome Amplification of Respiratory Syncytial Virus (RSV) using Illumina CovidSeq reagents for Next-Generation Sequencing V.3

DOI
dx.doi.org/10.17504/protocols.io.eq2lyjzbrlx9/v3
  • 1Microbiology and Infectology Department, IIS Galicia Sur;
  • 2Universidade de Vigo, Vigo, Spain;
  • 3Microbiology Department, Complexo Hospitalario Universitario de Vigo (CHUVI), Spain
Carlos Davina-Nunez
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lyjzbrlx9/v3
Protocol Citation: Carlos Davina-Nunez, Sonia Perez-Castro, Montse Godoy-Diz, Benito Regueiro-Garcia 2024. Whole-Genome Amplification of Respiratory Syncytial Virus (RSV) using Illumina CovidSeq reagents for Next-Generation Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjzbrlx9/v3Version created by Carlos Davina-Nunez
Manuscript citation:
Davina-Nunez, Carlos, Sonia Perez-Castro, Jorge Julio Cabrera-Alvargonzalez, Jhon Montano-Barrientos, Montse Godoy-Diz, and Benito Regueiro. "The Modification of the Illumina CovidSeq Workflow for RSV Genomic Surveillance: The Genetic Variability of RSV during the 2022–2023 Season in Northwest Spain." International Journal of Molecular Sciences 24, no. 22 (2023): 16055.
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: January 03, 2024
Last Modified: February 22, 2024
Protocol Integer ID: 92902
Keywords: Respiratory Syncytial Virus, RSV, NGS, Illumina, CovidSeq, amplification, PCR
Abstract
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.

Materials
ReagentQIASymphony DSP Virus/Pathogen Midi KitQiagenCatalog #937055
ReagentIllumina CovidSeq AssayIllumina, Inc.
ReagentQubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851 ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626

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).
Primer pools preparation
Primer pools preparation
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.
ABCDEF
PrimerVolume (100uM)SourceSequenceBasePool
A1f2,5WangACGSGAAAAAATGCGTACAAC11
A1r2,5WangGAAGATTGTGCTATACCAAAATGAACA17791
AB3f10GoyaGCYATGGCAAGACTYAGGAATG28971
A3r3,5WangGTTTGCYGAGGCTATGAATATGAT48261
A5f2,5WangGAACAACAGACTACTAGAGATTACCAG63741
A5r5This PublicationAGGAGTTTRCTCATRGCAA79291
A7f2,5WangAGCTTAGGCTTAAGATGYGGA94231
A7r2,5WangTGAGTTTGACCTTCCATGAGT109971
A9f5WangGGGTTGGTTCATCTACACAAGAG123161
A9r5This PublicationCGCAATAATAAATTCCCTGCTCC140941
B1f2WangACGCGAAAAAATGCGTACTACA11
B1r2WangCATTGTTTGCCCTCCTAATTACTG16611
B3r5WangATAGGGCCAAAATTTGCTTGTG43091
B5f2,5WangAGTGCAATCTTCCTAACTCTTGC57001
B5r2,5WangTGATTCCACTTAGTTGGTCTTTGC73751
B7f2,5WangGGTGAACTGAAATTAGAAGAACCAAC87601
B7r2,5WangCACCATATCTTGTCAAACTCTCAGG105071
B9f5WangGAACCAACTTACCCTCATGGATT118601
B9r5WangTTCTGGGGTTGGGTGATATAG136501
A2f2,5WangACAGGCATGACTCTCCTGAT15562
A2r2,5WangTTGGGTGTGGATATTTGTTTCAC34002
A4f2,5WangACCTGGGACACTCTCAATCA46972
A4r2,5WangGACATGATAGAGTAACTTTGCTGTCT65402
A6f2,5WangGTCACGAAGGAATCCTTGCA76422
A6r2,5WangCCCTCTACCTCTTTTATTATGTAGAACC95212
A8f5WangGGTGTACAATCTCTATTTTCCTGGT107042
A8r5WangCGATTAATAGGGCTAGTATCAAAGTG126152
A10f3,5This PublicationCCCACACCAGAAACCCTAGAAA136532
A10r3,5WangACGAGAAAAAAAGTGTCAAAAACTAA152252
B2f2WangCAGRTTAGGAAGGGAAGACACTA13162
B2r2WangCAAGTCACTCAATTTTTTGGAGGTTGG29822
B4f7,5WangTGGAAGCAYACAGCTACACG39432
B4r7,5WangCTACATGTYGATTGGTAAAACTCC57882
B6f2,5WangCCTCTAGTGTTTCCTTCTGATGAG71132
B6r2,5WangGTTGTAGCAATTTGTTCAGACGAG88342
B8f5WangAAGTTCTCTGAAAGCGACAGATC102312
B8r5PropioTAATACTWGGTGATGTTACTCCTAC121902
B10f2,5WangTAGTCAATCAAGACACAAGTTTGC132892
B10r2,5WangACGAGAAAAAAAGTGTCAAAAACTAATG152222
Table 1: mix of primers used for whole genome amplification. Two mixes are required, one for pool 1 and another for pool 2. References for base number: hRSV/A/England/397/2017 and hRSV/B/Australia/VIC-RCH056/2019 for RSV-A and RSV-B respectively. Citation to the source papers for the primers can be found below.

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.

ABCDEF
PrimerVolume (100uM)SourceSequenceBasePool
AB3f7,5GoyaGCYATGGCAAGACTYAGGAATG28971
A3r3,5WangGTTTGCYGAGGCTATGAATATGAT48261
A5f2,5WangGAACAACAGACTACTAGAGATTACCAG63741
A5r5This PublicationAGGAGTTTRCTCATRGCAA79291
B5f5WangAGTGCAATCTTCCTAACTCTTGC57001
B5r5WangTGATTCCACTTAGTTGGTCTTTGC73751
A4f5WangACCTGGGACACTCTCAATCA46972
A4r5WangGACATGATAGAGTAACTTTGCTGTCT65402
B4f7,5WangTGGAAGCAYACAGCTACACG39432
B4r7,5WangCTACATGTYGATTGGTAAAACTCC57882
B6f2,5WangCCTCTAGTGTTTCCTTCTGATGAG71132
B6r2,5WangGTTGTAGCAATTTGTTCAGACGAG88342
Table 2: mix of primers used for G and F protein amplification. Two mixes are required, one for pool 1 and another for pool 2. References for base number: hRSV/A/England/397/2017 and hRSV/B/Australia/VIC-RCH056/2019 for RSV-A and RSV-B respectively. Citation to the source papers for the primers can be found below.

RT-PCR
RT-PCR
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.

ReagentAmount (ul) Reaction 1Amount (ul) Reaction 2
IPM1515
FSM3.23.2
RVT11
Nuclease-Free Water3.63.6
Primer pool 1 (10uM)1.2-
Primer pool 2 (10uM)-1.2
Table 3: Master mixes required for amplification of the RSV genome. Reaction 1 targets odd-numbered amplicons while reaction 2 targets even-numbered amplicons.

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):

ABC
42º60 min
98º2 min
98º15 s35 cycles
63º7 min
4ºPAUSE
Table 4: Thermocycler program for RT-PCR. Indicate 25 ul as volume and heat lid at 99ºC.

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


Expected result

Figure 1: RT-PCR result with peaks expected around 2000 bps. Representative image of an Agilent bioanalyzer of the amplification products on the WG protocol. From left to right: ladder; RSV-A, pool 1; RSV-A, pool 2; RSV-B, pool 1; RSV-B, pool 2. Scale indicates size in base pairs.




Library preparation
Library preparation
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.
Expected results
Expected results
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).


Expected result
ABCDEFG
IDProtocolTypeCtReadsMapped% Mapped
1WGRSVA18,782572819583737,25
2WGRSVA16,032171559921845,69
3WGRSVA21,021661156332338,12
4WGRSVA18,325261015159160,01
5WGRSVA16,1518053710868360,20
6WGRSVA18,721518063918125,81
7WGRSVA16,561631035434633,32
8WGRSVA17,571984507592738,26
9WGRSVA19,791011881311412,96
10WGRSVA19,831853375695430,73
11WGRSVA19,541604655901936,78
12WGRSVB20,681955536504133,26
13WGRSVB20,531643142095012,75
14WGRSVB19,3327352112981347,46
15WGRSVB20,12168503827417,65
16WGRSVB19,771906893154016,54
17WGRSVB19,3240702712717,51
18WGRSVB15,7835248724822170,42
19WGRSVB19,111501375717238,08
20WGRSVB18,83152340142599,36
21WGRSVB17,7823744112888354,28
22WGRSVB19,1623141810610545,85
23WGRSVB18,981944175999730,86
24WGRSVB19,572369389330639,38
25GFRSVA24,5645961983130,70
26GFRSVA26,649369789916,00
27GFRSVA26,95969017312,90
28GFRSVA20,5473271324227,98
29GFRSVA20,83378512233,62
30GFRSVB21,65669055849,85
31GFRSVB24,36360717812,80
32GFRSVB22,96157757399,32
33GFRSVB26,56085514242,34
34GFRSVB22,6599491305721,78
Table 5: Results of sequenced samples in a NextSeq1000 run. 24 samples sequenced full genome and 10 sequences GF in the same run.

Figure 2: Coverage per base of all samples described in Table 3.






Bioinformatic Analysis
Bioinformatic Analysis
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.

References
References
The illumina CovidSeq protocol can be found in:

Illumina CovidSeq Reference Guide

The primers found in Table 1 were obtained from:

CITATION
Wang L, Ng TFF, Castro CJ, Marine RL, Magaña LC, Esona M, Peret TCT, Thornburg NJ (2022). Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes..
LINK
https://doi.org/10.1016/j.jviromet.2021.114335

CITATION
Stephanie Goya, Gabriel L. Rojo, Mercedes S. Nabaes Jordar, Laura E. Valinotto, Alicia S Mistchenko, Mariana Viegas. Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load. protocols.io.
LINK
https://protocols.io/view/whole-genome-sequencing-of-respiratory-syncytial-r-bmhak32e

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

CITATION
Ying Lin, Jeffrey Koble, Priyanka Prashar, Anita Pottekat, Christina Middle, Scott Kuersten, Michael Oberholzer, Robert Brazas, Darcy Whitlock, Robert Schlaberg, Gary P. Schroth. A sequencing and subtyping protocol for Influenza A and B viruses using Illumina® COVIDSeq™ Assay Kit. protocols.io.
LINK
https://protocols.io/view/a-sequencing-and-subtyping-protocol-for-influenza-crv3v68n