SARS-CoV-2 Genome Sequencing and ARTIC Network V3 Panel

    Viruses can constantly change their genome over time leading to the emergence of new variants with different characteristics. Sequencing technology enables to rapidly develop diagnostic tests and tools for outbreak management. Virus genome sequencing has already proven its potential in identifying the causative agents and their variants for COVID19 and investigate the pandemic outbreak dynamics, spatiotemporal spread, and transmission routes. It is also useful in designing strategies for diagnostic assays, drugs, and vaccines to improve public health. We offer SARS-CoV-2 whole-genome sequencing based on amplicon sequencing protocol developed by ARTIC network and Illumina NovaSeq 6000 system. We used the established methods for preparing amplicon pools in order to sequence SARS-CoV-2 genomes. ARTIC protocol includes a set of primers, laboratory protocols, bioinformatics tutorials, and datasets. ARTIC v3 primer sets improve the coverage in problematic amplicons by the addition of alternative primers (alts). It also reduce the cost and streamline the preparation of SARS-CoV-2 sequencing libraries. It would be helpful for clinical and research teams to obtain accurate and unbiased results using the ARTIC v3 panel.

 

Key requirements
•    Swab samples should be collected in viral transport medium (VTM)
•    The expedited shipping time is maximum 36 hours of sample collection
•    RNA extraction can be done using bead or column based methods
•    Turn Around Time will be 3-4 weeks
•    Library Type: cDNA library
•    RNA Integrity Number (Agilent 2100) ranges from 6-8
•    Purity (NanoDrop) – OD 260/280 = 1.8-2.2, OD 260/230 ≥ 1.8
•    Total RNA amount ≥ 0.4 µg
•    Amplified cDNA amount ≥ 100ng

 

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COVID-19 Disease and its origin and mutations
 
COVID-19 is a disease caused by the SARS-CoV-2 virus that involves acute respiratory complications. It is reported in 220 countries as of April 2021 after the identification of the first case originated in Wuhan, China in December 2019. The emergence of new variants and the multiple phenotypes generate new goals in understanding the dynamics of SARS-CoV-2 infectivity and transmissibility. SARS-CoV-2 genome is rapidly undergoing genetic mutations. Phylogenetic analysis reveals hundreds of variants documented till today.
  • Denmark reported the ‘Cluster 5’ variant was linked to infection among farmed mink that was subsequently blocked by culling 17 million minks in November 2020.
  • United Kingdom reported a variant referred to as SARS-CoV-2 VOC 202012/01. More than 60 countries have detected UK variants in samples of positive cases.
  • South Africa announced the detection of a new variant 50 1Y.V2 on 18 December 2020, which is rapidly spreading in three provinces of South Africa.
  • United States reported B.1.1.7 and B.1.351 which was first identified in the US but initially detected in UK and South Africa respectively, P.1, B.1.427 and B.1.429 were classified as Variant of Concern (VOCs) in March 2021.
  • India reported B.1.617 variant as a dominant strain in the second wave and spread to about 40 other nations. The lineage B.1.617 was defined by the mutations E484Q, L452R, and P681R, D614G with additional mutation Q1071H in the spike protein. The Indian origin B.1.617.2 was detected in December 2020 with additional mutations namely T19R, DEL157/158, T478K, and D950N. B.1.617.3 variant has the following additional mutations: T19R, DEL157/158 and D950N. Whereas epidemiologists perform risk assessment for different variants to determine the public health implications, researchers are routinely assessing genomics data to investigate the impact of SARS-CoV-2 variants on virus transmissibility, disease severity, the efficacy of diagnostics, therapeutics and vaccines.