An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates

Goya, Stephanie; Valinotto, Laura; Tittarelli, Estefanía; Rojo, Gabriel Lihue; Jodar, Mercedes Soledad Nabaes; Greninger, Alexander L.; Zaiat, Jonathan; Martí, Marcelo A.; Mistchenko, Alicia; Viegas, Mariana

doi:10.1371/journal.pone.0199714

Cited by 30 publications

(33 citation statements)

References 39 publications

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“…While the number of target reads increased with the amount of data generated, the removal of a greater proportion of duplicate reads led to lower proportions of target reads in Illumina MiSeq 500 v2 runs (MK5, MN5). A similar finding was reported in a study optimizing methodologies for sequencing of human respiratory syncytial virus, with higher proportions of duplicate reads observed in the higher output Illumina NextSeq 500 datasets compared to the MiSeq (35). This is most likely due to over-amplification of viral genomes during SISPA, combined with a greater probability with increasing sequencing depth of generating duplicate reads by chance, especially for small genomes (36).…”

Section: Discussionsupporting

confidence: 84%

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Marine¹,

Valladares

Castro

et al. 2019

Preprint

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28Next-generation sequencing is a powerful tool for virological surveillance. While 29 Illumina® and Ion Torrent® sequencing platforms are used extensively for generating 30 viral RNA genome sequences, there is limited data comparing different platforms. We 31 evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a 32 panel of sixteen specimens containing picornaviruses and human caliciviruses 33 (noroviruses and sapoviruses). The specimens were processed, using combinations of 34 three library preparation and five sequencing kits, to assess the quality and 35 completeness of assembled viral genomes, and an estimation of cost per sample to 36 generate the data was calculated. The choice of library preparation kit and sequencing 37 platform was found to impact the breadth of genome coverage and accuracy of 38 consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM 39 platform in data quality and cost, and generated the highest proportion of reads for 40 enterovirus D68 samples. However, indels at homopolymer regions impacted the 41 accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., 42 Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the 43 MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq 44 V2) the cost per sample was comparable. These findings suggest that the Ion Torrent 45 S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of 46 RNA viruses, each with specific advantages and tradeoffs.47 48Conventional Sanger sequencing has been the gold standard for genomic analysis of 49 pathogens in public health laboratories for over three decades. However, the expansion 50 of next-generation sequencing (NGS) technologies has increased demand for high-51 throughput sequencing of genomes at a lower cost (1). NGS has been used extensively 52 for routine surveillance and outbreak investigation of numerous viral RNA pathogens. 53The exponential growth of genomic information generated for important pathogens has 54 provided increased resolution for molecular epidemiology, as well as information 55 necessary for the design of clinical assays and therapeutics (2-5). NGS methods are 56 also useful for identifying pathogens in syndromes where etiologies often remain 57 unknown (e.g., encephalitis, febrile illness), complementing or even replacing current 58 diagnostic methods (2, 6, 7). 60Over the past several years, the suppliers of high-capacity short-read sequencers have 61 been reduced to two manufacturers: Illumina (sequencing-by-synthesis technology) and 62 Thermo Fisher Scientific (Ion Torrent semi-conductor sequencing technology) (3). 63 Illumina platforms have been used to generate nearly 90% of NGS data worldwide 64 (https://www.wired.com/2016/02/gene-sequencing-goliath-wants-get-bigger-still/). 65Illumina produces several benchtop and production-scale sequencers with data outputs 66 varying from 1.2 gigabases (Gb) to 6 terabases (Tb). In mic...

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Section: Discussionsupporting

confidence: 84%

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Marine¹,

Valladares

Castro

et al. 2019

Preprint

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“…One study performed in 2008 showed that the NA1 strain was the predominant genotype (90.9%) in the Riyadh region with no incidence of the ON1 strain [41], while the other study [6], also performed in Riyadh, in 2012, showed the first incidence of the ON1 genotype, but the NA1 genotype was still the predominant genotype (82.6%). Saudi strains from Taif (2019), reported by Al Aboud et al [42], for samples collected in 2012, belong to the NA1 genotype (6/13, 46.15%), ON1 (5/13; 38.46%) and GA5 genotype (2/13; 15.38%). Recent sequences retrieved from GenBank (accession numbers MH388029 to MH388042) for samples collected from Riyadh, Saudi Arabia, between 2014 and 2016, showed 14 RSV-A samples of the ON1 type, but the work was not published, and we have no prevalence data on this study.…”

Section: Discussionmentioning

confidence: 96%

Dominance of the ON1 Genotype of RSV-A and BA9 Genotype of RSV-B in Respiratory Cases from Jeddah, Saudi Arabia

Al-Sharif

El‐Kafrawy

Yousef

et al. 2020

Genes

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Human respiratory syncytial virus (HRSV) is a main cause of hospital admission for lower respiratory tract infection. In previous studies from Saudi Arabia, higher prevalence of the NA1 genotype in group A was observed from Riyadh and Taif. This study recruited respiratory cases from Jeddah during January to December, 2017. RSV represented 13.4% in the recruited cases with 64% of them belonging to group A and 36% to group B. All group A cases in this study were ON1 type characterized by duplication of 72 nucleotides, 24 amino acids in the C-terminal in the second hypervariable region of the G gene. In addition, for group B all of the cases were clustered under BA9, which had uniquely characterized as duplication of 60 nucleotides in the G protein. Our sequences showed similarity with earlier sequences from Saudi Arabia, Kuwait, Thailand, South Africa, Spain, the USA and Cyprus. Some amino acid substitutions in the investigated sequences would cause a change in potential O-glycosylation and N-glycosylation profiles from prototype ON1. The predominance of the ON1 and BA9 genotype of RSV-A in Jeddah compared to previous Saudi studies showing predominance of the NA1 genotype for group A. This difference in genotype prevalence could be due to fast spread of the ON1 genotype worldwide or due to the flux of travelers through Jeddah during hajj/umrah compared to Riyadh and Taif. This shift in genotype distribution requires continuous surveillance for genetic characterization of circulating respiratory infections including RSV. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.

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“…To understand the diversity of strains circulating in the community, samples collected from patients at facilities within the University of Washington (UW) medical system, including SCH patients with community-acquired norovirus, were also sequenced (Supplementary Table S1). mNGS sequencing was performed as described previously [12,16] (see Supplementary Methods).…”

Section: Methodsmentioning

confidence: 99%

Prospective real-time metagenomic sequencing during norovirus outbreak reveals discrete transmission clusters

Casto

Adler

Makhsous

et al. 2018

Preprint

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8Background: Norovirus outbreaks in hospital settings are a common challenge for infection 3 9 prevention teams. Given the high burden of norovirus in most communities, it can be difficult to 4 0 distinguish between on-going in-hospital transmission of virus and new introductions from the 4 1 community and challenging to understand the long-term impacts of outbreak-associated viruses 4 2 within medical systems using traditional epidemiological approaches alone. 4 3 Methods: Real-time metagenomic sequencing during an on-going norovirus outbreak 4 4 associated with a retrospective cohort study. 4 5 Results:We describe a hospital-associated norovirus outbreak that affected 13 patients over a 4 6

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An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates

Cited by 30 publications

References 39 publications

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Dominance of the ON1 Genotype of RSV-A and BA9 Genotype of RSV-B in Respiratory Cases from Jeddah, Saudi Arabia

Prospective real-time metagenomic sequencing during norovirus outbreak reveals discrete transmission clusters

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