Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

Doddapaneni, Harshavardhan; Cregeen, Sara Javornik; Sucgang, Richard; Meng, Qingchang; Qin, Xiang; Avadhanula, Vasanthi; Chao, Hsu; Menon, Vipin; Nicholson, Erin; Henke, David; Piedra, Felipe-Andrés; Rajan, Anubama; Momin, Zeineen; Kottapalli, Kavya; Hoffman, Kristi L.; Sedlazeck, Fritz J.; Metcalf, Ginger; Piedra, Pedro A.; Petrosino, Joseph F.; Gibbs, Richard A.

doi:10.1371/journal.pone.0244468

Cited by 24 publications

(15 citation statements)

References 41 publications

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“…Previous studies have reported successful genome recovery in samples with low Ct values (<25) ( 28 , 29 ). Our findings indicated that there was no significant correlation between genome coverage and Ct value when using either version of the ARTIC primers.…”

Section: Discussionmentioning

confidence: 99%

“…Our findings indicated that there was no significant correlation between genome coverage and Ct value when using either version of the ARTIC primers. Genomes with >95% coverage were recovered from samples with higher Ct values ( 24 – 29 ), and the differences observed could be either due to sample-to-sample variation or batch processing. Therefore, when using the optimized V4 primers, genome completeness (>95%) can be expected for samples with a wide Ct value range ( 14 – 29 ) regardless of the lineage.…”

Section: Discussionmentioning

confidence: 99%

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Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

et al. 2022

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IntroductionThe ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs.MethodsWe utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'.ResultsWe observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery.ConclusionUtilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

et al. 2022

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“…Shotgun sequencing requires no prior knowledge of the targeted viral genome 15 but is limited by requirements for a high viral load and a higher sequencing depth. Hybridization approaches target genomic regions of interest by using biotinylated probes and ensure a more complete profiling of regions of interest 16 . Since the initial release of the ARTIC SARS-CoV-2 sequencing protocol early in the outbreak (Jan 22, 2020, https://artic.network/ncov-2019 ), amplicon-based sequencing has become the primary choice for many labs around the world and numerous commercial kits are also available 14 .…”

Section: Introductionmentioning

confidence: 99%

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory

Rosenthal

Герасимова

Ruiz-Vega

et al. 2022

Sci Rep

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Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.

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“…(2) A genotyping PCR panel could be very useful in resource-limited settings, since sequencing is not done at the same level in all locations/countries, and many laboratories do not have the infrastructure or expertise to perform sequencing ( 51 ). (3) Obtaining whole genome sequence data from clinical samples is very much dependent on viral load; reported PCR Cq cutoffs for obtaining full-length genome sequences range from ~25–33 ( 52 , 53 ). A PCR panel could fill this gap by providing genotype information for numerous samples that cannot be sequenced due to low viral load as reflected by high Cq values ( 31 ).…”

Section: Discussionmentioning

confidence: 99%

Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform

Stanhope¹,

Peterson

Knight

et al. 2022

Front. Public Health

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Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69–70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1–BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays exhibit high sensitivity and specificity. This panel can help fulfill the need for rapid identification of variants in samples, leading to quick decision making with respect to public health measures, as well as treatment options for individuals. Compared to sequencing, these genotyping PCR assays allow much faster turn-around time from sample to results—just a couple hours instead of days or weeks.

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Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

Cited by 24 publications

References 41 publications

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory

Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform

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