A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

Gohl, Daryl M.; Garbe, John; Grady, Patrick G. S.; Daniel, Jerry; Watson, Ray H. B.; Auch, Benjamin; Nelson, Andrew C.; Yohe, Sophia; Beckman, Kenneth B.

doi:10.1101/2020.05.11.088724

Cited by 14 publications

(23 citation statements)

References 21 publications

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“…We adopted amplicon-based sequencing (amplicon-seq), a method widely used to characterize SARS-CoV-2 genomes 25 , to characterize the presence of sgRNAs and correlate their abundance in the COVID-19 positive samples between symptomatic and asymptomatic patients. Ampliconseq is highly sensitive, with limit of detection (LoD) reported as low as one SARS-CoV-2 copy per microliter using the optimized protocols from the Artic network 26,27 . Therefore, it can effectively enrich for SARS-CoV-2 cDNAs from samples of wide-range of viral content.…”

Section: Sgrna Expression Is Drastically Repressed In Asymptomatic Samentioning

confidence: 99%

Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection

Wong

Ngan

Goldfeder

et al. 2021

Preprint

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In coronaviridae such as SARS-CoV-2, subgenomic RNAs (sgRNA) are replicative intermediates, therefore, their abundance and structures could infer viral replication activity and severity of host infection. Here, we systematically characterized the sgRNA expression and their structural variation in 81 clinical specimens collected from symptomatic and asymptomatic individuals with a goal of assessing viral genomic signatures of disease severity. We demonstrated the highly coordinated and consistent expression of sgRNAs from individuals with robust infections that results in symptoms, and found their expression is significantly repressed in the asymptomatic infections, indicating that the ratio of sgRNAs to genomic RNA (sgRNA/gRNA) is highly correlated with the severity of the disease. Using long read sequencing technologies to characterize full-length sgRNA structures, we also observed widespread deletions in viral RNAs, and identified unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Furthermore, based on the sgRNA structures, the frequently occurred structural variants in SARS-CoV-2 genomes serves as a mechanism to further induce SARS-CoV-2 proteome complexity. Taken together, our results show that differential sgRNA expression and structural mutational burden both appear to be correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

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Section: Sgrna Expression Is Drastically Repressed In Asymptomatic Samentioning

confidence: 99%

Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection

Wong

Ngan

Goldfeder

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…Several approaches have been used to sequence the SARS-CoV-2 genome, including metagenomics (Manning et al 2020) , sequence capture (Gohl et al 2020) , SISPA (Moore et al 2020) , and multiplex PCR (Gohl et al 2020;Itokawa et al 2020;Resende et al 2020;Moore et al 2020;Eden et al 2020) , followed by next generation sequencing using either the Illumina or Oxford Nanopore platforms. Due to its simplicity and economy, using multiplexed PCR amplicons is perhaps the most common approach.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

Freed

Vlková

Faisal

et al. 2020

Preprint

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Rapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with C q values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

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“…In a recent paper, Kim et al [3] explored the transcriptomic architecture of SARS-CoV-2 by performing short-read as well as long-read sequencing of Vero cells infected by the virus. The authors used oligo(dT) amplification, which targets the poly(A) tail at the 3’ end of messenger RNAs, thus limiting positional bias that would occur when using SARS-CoV-2 specific primers [25, 26]. Subsequently, the authors aligned the resulting reads using splice-aware aligners, i.e.…”

Section: Resultsmentioning

confidence: 99%

Jumper Enables Discontinuous Transcript Assembly in Coronaviruses

Sashittal

Zhang

Peng

et al. 2021

Preprint

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Genes in SARS-CoV-2 and, more generally, in viruses in the order of Nidovirales are expressed by a process of discontinuous transcription mediated by the viral RNA-dependent RNA polymerase. This process is distinct from alternative splicing in eukaryotes, rendering current transcript assembly methods unsuitable to Nidovirales sequencing samples. Here, we introduce the DISCONTINUOUS TRANSCRIPT ASSEMBLY problem of finding transcripts T and their abundances c given an alignment R under a maximum likelihood model that accounts for varying transcript lengths. Underpinning our approach is the concept of a segment graph, a directed acyclic graph that, distinct from the splice graph used to characterize alternative splicing, has a unique Hamiltonian path. We provide a compact characterization of solutions as subsets of non-overlapping edges in this graph, enabling the formulation of an efficient mixed integer linear program. We show using simulations that our method, JUMPER, drastically outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1 and SARS-CoV-2 samples, we find that JUMPER not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are well supported by direct evidence from long-read data, presence in multiple, independent samples or a conserved core sequence. JUMPER enables detailed analyses of Nidovirales transcriptomes.

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A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

Cited by 14 publications

References 21 publications

Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection

Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection

Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

Jumper Enables Discontinuous Transcript Assembly in Coronaviruses

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