SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms

Abstract

Genomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.

Genomic surveillance of SARS-CoV-2 reveals community transmission of a major lineage during the early pandemic phase in Brazil

“…In this study, we analyzed 95 viral whole-genomes (>99% coverage) obtained from individuals with confirmed SARS-CoV-2 infection, who underwent testing and genomic sequencing at the Laboratory of Respiratory Viruses and Measles, Oswaldo Cruz Institute (IOC)-FIOCRUZ, in Rio de Janeiro, and the Evandro Chagas Institute, in Para, Brazil 25 . Samples were collected between 29 th February and 28 th April 2020 from individuals that reside in 10 different Brazilian states from the Southeastern (Rio de Janeiro and Espirito Santo), Central-Western (Distrito Federal), Northern (Acre, Amapa and Para), Northeastern (Alagoas, Bahia and Maranhao) and Southern (Santa Catarina) regions.…”

“…Total RNA from positive samples presenting Ct values up to 30,0 for gene E was reverse transcribed using SuperScript™ IV First Strand Synthesis System (Invitrogen). Two multiplex PCR reactions using the primer scheme previously described 25 (Pool A = nine amplicons and Pool B = eight amplicons), were performed using the Q5® High-Fidelity DNA Polymerase (NEB). Amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter™) and the DNA quantified with Qubit 4 Fluorometer (Invitrogen) using the Qubit dsDNA HS Assay Kit (Invitrogen) and sequenced using Illumina MiSeq or NextSeq (San Diego, CA, USA) and Nanopore (Oxford, UK) platforms.…”

“…The size distribution of these libraries was evaluated using a 2100 Bioanalyzer (Agilent, Santa Clara, USA) and the samples were pair-end sequenced (Micro V2, 300 cycles) on a MiSeq equipment (Illumina, San Diego, USA) in around 18 hours. The Nanopore library protocol is optimized for long reads (2 kb amplicons) 25 . Library preparation was conducted using Ligation Sequencing 1D (SQK-LSK109 Oxford Nanopore Technologies (ONT) and Native Barcoding kit 1 to 24 (ONT), according to the manufacturer’s instructions.…”

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

“…Other groups have proposed alternate primer schemes using longer amplicons to improve genome coverage [25,26] . Our primer scheme uses 400 bp amplicons which are compatible with both long and short-read sequencing platforms and offer good performance with degraded or high Ct samples.…”

Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

“…Several approaches have been used to sequence the SARS-CoV-2 genome, including metagenomics (Manning et al 2020) , sequence capture (Gohl et al 2020) , SISPA (Moore et al 2020) , and multiplex PCR (Gohl et al 2020;Itokawa et al 2020;Resende et al 2020;Moore et al 2020;Eden et al 2020) , followed by next generation sequencing using either the Illumina or Oxford Nanopore platforms. Due to its simplicity and economy, using multiplexed PCR amplicons is perhaps the most common approach.…”