5/10/2023 0 Comments Oxford nanopore midnight protocol![]() The V3 primer were released on 24th March 2020 together with an improved overall sequencing approach ( 13). Subsequently, an improved set of ARTIC V2 primers were released. The ARTIC V1 protocol and primer-set had a number of challenges including drop-offs at amplicons 18 and 76 due to primer dimers ( 17). The first version of this protocol was released to the public on 22nd January 2020 and comprised of what became the ARTIC V1 primer-set that consisted of 98 primer pairs spanning the ~30kb except for the 3' and 5' regions. ![]() The protocol had five key steps (i) cDNA synthesis using superscript IV kit, (ii) multiplex RT-PCR using Q5 kit and ARTIC V1 primers in two pools, (iii) RT-PCR clean-up using beads, quantification and normalization, (iv) native barcode ligation and (v) sequencing on the MINION device. It employed an early draft version of the SARS-CoV-2 genome and incorporated two sets of primer pools for efficient multiplexing ( 15, 16). This protocol was developed based on an earlier strategy for sequencing single-stranded RNA viruses from high cycle threshold (Ct) clinical samples ( 14). The most widely adopted targeted amplicon approach for SARS-CoV-2 genomic sequencing is the ARTIC protocol. Amplicon based methods using SARS-CoV-2 specific primers that amplify between 400 to 2,500 base pairs were designed and implemented using multiplex RT-PCR methods followed by WGS using platforms such as Oxford Nanopore Technologies and Illumina ( 11– 13). Early SARS-CoV-2 genomes were generated using a metagenomic approach given the lack of reference genome at the beginning of the pandemic ( 9). There are several approaches used for whole genome sequencing (WGS) of SARS-CoV-2 and can be broadly categorized as targeted and non-targeted i.e., metagenomic approaches ( 9– 12). SARS-CoV-2 genomes help explain virus evolution and transmission ( 4, 5), identify sites on the genome that may aid vaccine/antibody evasion and inform vaccine design ( 6), improve design of molecular and serological assays ( 7) and influence public health policy ( 8). Genomic sequencing of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been instrumental in understanding the biology, emergence and spread of the virus globally ( 1– 3). However, only 60–70% of the genomes could be recovered in samples that had 0.05) correlation between Ct value and genome recovery.Ĭonclusion: Utilizing 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. Consequently, ≥95% of the genome was recovered in 72% ( n = 31) of the samples. 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. ![]() Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. Results: We 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. Methods: We 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'. 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. 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. Introduction: The ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol.
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