{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Kandel S"],"funding":["NCATS NIH HHS","NIGMS NIH HHS"],"pagination":["1272972"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10910555"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["15"],"pubmed_abstract":["Introduction
Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs).Methods
In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell.Results and discussion
Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants."],"journal":["Frontiers in microbiology"],"pubmed_title":["Genomic surveillance of SARS-CoV-2 using long-range PCR primers."],"pmcid":["PMC10910555"],"funding_grant_id":["UL1 TR003107","P20 GM121293"],"pubmed_authors":["Ingold AK","Kennedy JL","Ussery DW","Kandel S","Turner GA","Hartzell SL"],"additional_accession":[]},"is_claimable":false,"name":"Genomic surveillance of SARS-CoV-2 using long-range PCR primers.","description":"Introduction
Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs).Methods
In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell.Results and discussion
Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.","dates":{"release":"2024-01-01T00:00:00Z","publication":"2024","modification":"2026-07-01T03:24:15.815Z","creation":"2025-04-19T22:01:06.585Z"},"accession":"S-EPMC10910555","cross_references":{"pubmed":["38440140"],"doi":["10.3389/fmicb.2024.1272972"]}}