7 research outputs found
Genomic surveillance of SARS-CoV-2 using long-range PCR primers
IntroductionWhole 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).MethodsIn 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 discussionUsing 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
Anthrax outbreak in a Swedish beef cattle herd - 1st case in 27 years: Case report
After 27 years with no detected cases, an outbreak of anthrax occurred in a beef cattle herd in the south of Sweden. The outbreak was unusual as it occurred in winter, in animals not exposed to meat-and-bone meal, in a non-endemic country
Image_1_Genomic surveillance of SARS-CoV-2 using long-range PCR primers.TIFF
IntroductionWhole 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).MethodsIn 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 discussionUsing 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.</p
Table_1_Genomic surveillance of SARS-CoV-2 using long-range PCR primers.docx
IntroductionWhole 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).MethodsIn 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 discussionUsing 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.</p
Image_2_Genomic surveillance of SARS-CoV-2 using long-range PCR primers.TIF
IntroductionWhole 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).MethodsIn 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 discussionUsing 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.</p
Image_3_Genomic surveillance of SARS-CoV-2 using long-range PCR primers.TIF
IntroductionWhole 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).MethodsIn 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 discussionUsing 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.</p