SARS-CoV-2 Genome Sequencing Methods Differ in Their Abilities To Detect Variants from Low-Viral-Load Samples

Lam, Connie; Gray, Karen-Ann; Gall, Mailie; Sadsad, Rosemarie; Arnott, Alicia; Johnson-Mackinnon, Jessica; Fong, Winkie; Basile, Kerri; Kok, Jen; Dwyer, Dominic E.; Sintchenko,; Rockett, Rebecca

doi:10.1128/jcm.01046-21

Cited by 41 publications

(40 citation statements)

References 39 publications

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“…The SARS-CoV-2 viral RNA was detected and quantified using a previously described RT-qPCR targeting the N-gene ( Rahman et al, 2020 ; Lam et al, 2021 ). Ten-fold serial dilutions (1 × 10 6 to 1 × 10 2 copies/μL) of the commercially available synthetic RNA control reference strain (Wuhan-1 strain, TWIST Biosciences) containing six non-overlapping fragments of the SARS-CoV-2 reference sequence (NCBI GenBank accession MN908947.3 ) was used to generate a standard curve and quantify SARS-CoV-2 viral load.…”

Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture

et al. 2022

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BackgroundLow frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model.MethodsTwo clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33).ResultsIntrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts.Discussion and ConclusionThe positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

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Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture

et al. 2022

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“…Two respiratory swabs collected from Case A on Days 2 and 3, as well as two respiratory swabs from Case B collected on Days 3 and 11, were subjected to nucleic acid extraction, quantitative SARS-CoV-2 PCR and genome sequencing using short-read (NextSeq 500 (Illumina)) and long-read (GridION (Oxford Nanopore Technologies; ONT)) protocols using Midnight primers. We also employed the probe-based Illumina Respiratory Viral Oligo panel (RVOP) 9 to collect reads unbiased by SARS-CoV-2 PCR amplification (see Online Methods for details). Viral yield in samples was variable but still significant and suggesting the presence of viable virus (Table 1).…”

Section: Mainmentioning

confidence: 99%

Co-infection with SARS-COV-2 Omicron and Delta Variants Revealed by Genomic Surveillance

Rockett

Draper

Gall

et al. 2022

Preprint

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We identified the co-infection of the SARS-CoV-2 Omicron and Delta variants in two epidemiologically unrelated patients with chronic kidney disease requiring haemodialysis. Both SARS-CoV-2 variants were co-circulating locally at the time of detection. Amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified Omicron and Delta subpopulations in respiratory samples from the two patients. These findings highlight the importance of genomic surveillance in vulnerable populations.

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“…Blood and fecal specimens were only applied when the oropharyngeal region was injured or the patient cannot tolerate the swab collections. However, we must admit that blood and fecal specimens may result in the missing of low viral load patients because of the relatively low detection rates of these two specimens ( Lam et al., 2021 ). Therefore, our cohort is more likely to represent typical HAdV cases.…”

Section: Discussionmentioning

confidence: 99%

Clinical and Immunological Characteristics of Patients With Adenovirus Infection at Different Altitude Areas in Tibet, China

Wang

Peng

et al. 2021

Front. Cell. Infect. Microbiol.

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BackgroundThe severities of human adenovirus (HAdV) infection are diverse in different areas of Tibet, China, where a large altitude span emerges. Serious consequences may be caused by medical staff if the clinical stages and immunological conditions of patients in high-altitude areas are misjudged. However, the clinical symptoms, immunological characteristics, and environmental factors of HAdV infection patients at different altitude areas have not been well described.MethodsIn this retrospective, multicenter cohort study, we analyzed the data of patients who were confirmed HAdV infection by PCR tests in the General Hospital of Tibet Military Command or CDC (the Center for Disease Control and Prevention) of Tibet Military Command from January 1, 2019, to December 31, 2020. Demographic, clinical, laboratory, radiological, and epidemiological data were collected from medical records system and compared among different altitude areas. The inflammatory cytokines as well as the subsets of monocytes and regulatory T cells of patients were also obtained and analyzed in this study.ResultsSix hundred eighty-six patients had been identified by laboratory-confirmed HAdV infection, including the low-altitude group (n = 62), medium-altitude group (n = 206), high-altitude group (n = 230), and ultra-high-altitude group (n = 188). Referring to the environmental factors regression analysis, altitude and relative humidity were tightly associated with the number of infected patients (P < 0.01). A higher incidence rate of general pneumonia (45.7%) or severe pneumonia (8.0%) occurred in the ultra-high-altitude group (P < 0.05). The incubation period, serial interval, course of the disease, and PCR-positive duration were prolonged to various extents compared with the low-altitude group (P < 0.05). Different from those in low-altitude areas, the levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, TNF-α, TNF-β, and VEGF in the plasma of the ultra-high-altitude group were increased (P < 0.05), while the proportion of non-classical monocytes and regulatory T cells was decreased (P < 0.05).ConclusionsThe findings of this research indicated that patients with HAdV infection in high-altitude areas had severe clinical symptoms and a prolonged course of disease. During clinical works, much more attention should be paid to observe the changes in their immunological conditions. Quarantine of patients in high-altitude areas should be appropriately extended to block virus shedding.

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SARS-CoV-2 Genome Sequencing Methods Differ in Their Abilities To Detect Variants from Low-Viral-Load Samples

Cited by 41 publications

References 39 publications

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture

Co-infection with SARS-COV-2 Omicron and Delta Variants Revealed by Genomic Surveillance

Clinical and Immunological Characteristics of Patients With Adenovirus Infection at Different Altitude Areas in Tibet, China

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