DOI: 10.5061/dryad.cnp5hqc9v
title: Raw signals of nucleotides in the S gene of 12 SARS-COV-2 strains

description: The surveillance of the SARS-CoV-2 genome has become one of the crucial
 techniques in the management of COVID-19, aiding the pandemic response and
 supporting effective public health interventions. Typically, whole genomic
 sequencing is used along with PCR-based target enrichment techniques to
 identify SARS-CoV-2 variants, which is a complicated and time-consuming
 process that requires central laboratory facilities. Thus, there is an
 urgent requirement for developing rapid and cost-effective tools that can
 precisely detect and identify SARS-CoV-2 strains on-site. In this study,
 we demonstrate the diagnosis of COVID-19 patients and rapid identification
 of SARS-CoV-2 variants by amplifying and sequencing the entire length of
 the SARS-CoV-2 S gene using an isothermal enzymatic recombinase
 amplification combined with the most advanced Oxford nanopore sequencing.
 The entire procedure, from sampling to sequencing, takes less than 8 hours
 and can be performed with limited resources. The newly developed method
 has noteworthy implications for examining the transmission dynamics of the
 virus, detecting novel genetic variants, and assessing how mutations
 affect the efficiency of diagnostic approaches, antiviral treatments, and
 vaccines.
description: <strong>1/ Sampling and RNA
 extraction</strong> SARS-CoV-2 strains were
 isolated from the positive COVID-19 samples and used to extract genomic
 RNA using the QIAamp Viral RNA Mini Kit (Qiagen, Germany).
 <br><strong>2/ Primer design and colorimetric triplex
 RT-ERA assay</strong> The ERA primers
 were designed to amplify the entire sequence of the SARS-CoV-2 S gene
 based on the complete genome of the Wuhan-Hu-1 SARS-CoV-2 strain (NCBI
 Reference Sequence: MN908947.3). The length of all primers was optimized
 to be between 28-30 bp, producing amplicons ranging from 300-500 bp, which
 is suitable for ERA. A total of 12 primer pairs were designed to amplify
 the full region of the SARS-CoV-2 S gene. The
 reaction mixture (15 μl) was prepared in a 0.2 ml tube, consisting of 1.5
 μl primer mixture, 6 μl of the ERA buffer, 1 μl of template, and 6.3 μl of
 water. The reactions were incubated at 39°C for 40 minutes in a heat
 block.  <br><strong>3/
 Sequencing and bioinformatic analysis</strong> 
 The RT-ERA products for each sample were pooled and purified
 using the Monarch® PCR & DNA Cleanup Kit (NEB, UK). The 12 pooled
 samples were sequenced using Native Barcoding Expansion 1-12 (EXP-NBD104,
 ONT), the ligation sequencing kit SQK-LSK109 (ONT), and flow cell R9.4.1
 with MinION Mk1B device according to the manufacturer’s instruction. Data
 acquisition and real-time base-calling and data acquisition were carried
 out using the MinKNOW software and Nanopore Guppy tool,
 respectively. The obtained raw reads were filtered
 using the BBDuk tool and Geneious Prime version 2020.2.2. The clean
 sequences were then assembled to the S genes using the Map to Reference
 function in Geneious Prime. The final sequence was annotated using the
 Nextclade v2.11.0 website (https://clades.nextstrain.org/), which provides
 information on the SARS-CoV-2 variant.
description: The raw data can be read using Guppy Basecaller
 (https://github.com/asadprodhan/GPU-accelerated-guppy-basecalling). Then,
 fastq files will be generated and used for further analysis steps such as
 quality check, assembly, comparative analysis.