NGS-SA: Genomic and Bioinformatic protocols to SARS-CoV-2

NEBnext library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol

Title: NEBnext library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol

Authors: Giandhari J, Pillay S, Tegally H, Wilkinson E, Chimukangara B, Lessells R, Moosa Y, Gazy I, Fish M, Singh L, Khanyile KS, Fonseca V, Giovanetti M, Alcantara LC, de Oliveira T

Date: 2020-07-26

Abstract

Introduction

This protocol describes a method for whole genome sequencing of the SARS-CoV-2 using a tiling PCR approach with overlapping primers and NEBNext Ultra II Library Preparation Kit for Illumina sequencers. This method was produced in KRISP labs for the Network of Genomics Surveillance of South Africa (NGS-SA).

Briefly, primers are designed to be 20-30bp in length and to generate 400bp amplicons with a 70bp overlap. The primers are designed using an online tool called Primal Scheme (http://primal.zibraproject.org/).The amplicons generated can be sequenced on the on the Illumina MiSeq. This will produce next generation sequences covering the whole genome of the SARS-CoV-2 .

Purpose

The purpose of this document is to provide detailed instructions that should be followed when performing the sequencing of SARS-CoV-2 whole genomes from RNA samples using the NEBNext Ultra II Library Preparation Kit.


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