Raw signals of nucleotides in the S gene of 12 SARS-COV-2 strains
Data files
Aug 01, 2023 version files 241.27 MB
Abstract
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.
Methods
1/ Sampling and RNA extraction
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).
2/ Primer design and colorimetric triplex RT-ERA assay
- 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.
3/ Sequencing and bioinformatic analysis
- 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.
Usage notes
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.