SARS-CoV-2 surveillance: Nanopore sequencing as potential rapid tool for novel pathogen variants characterization

ABSTRACT

Genomic epidemiology is a crucial weapon in the public health fight against infectious diseases.Whole- Genome Sequencing (WGS) of SARS-CoV-2 provides critical insight into viral transmission and evolution.Oxford Nanopore Technology (ONT) works by monitoring changes to an electrical current as a strand of nucleic acid passes through a protein nanopore and the resulting signal from multiple nucleotides is decoded to provide the specific sequence.Illumina sequencing technology, sequencing by synthesis (SBS), detects base-by-base as they are incorporated into growing DNA strands enabling accurate data. In the present study a comparison between ONT performance on MinION and Illumina ISeq was evaluated.Library preparation was performed on 25 SARS-CoV-2-positive specimens collected at Hygiene Laboratory, San Martino Hospital (Genoa, Italy) by COVID panel CE-IVD kit developed at 4Bases and CleanPlex SARS-CoV-2 NGS Panel by Paragon Genomics, respectively to be sequenced on ONT MinION MK1B and Illumina iSeq100 platforms.Data analysis was carried out using the 4eVAR (4Bases) and Sophia DDM Software (Sophia Genetics) platforms and consensus sequence was analyzed using Nextclade webservice. Each variant was classified according to World Health Organization (WHO), Clade and Lineage.Both technologies correctly identified 7 Delta and 18 Omicron variants. Mean coverage on MinION was 763x, after 3 hours of run, covering 98% of the genome. There was perfect match for the WHO, Clade and the Lineage classification except for one difference along the sub-lineage classification of one omicron variant (BA.2 in MinION, BA.2.3 in Paragon).Single mutations analysis showed 96% overlap on average in mutation classification considering all mutations, 98% considering mutations on Spike protein.The two different technologies show a comparable analytical performance, with small differences concerning precise mutation classification. MinION enables real-time analysis, therefore is faster, cheaper and more flexible than standard sequencing techniques. By this analysis it is confirmed to be a promising platform, with the potential to represent a convenient portable and rapid tool for the SARS-COV2 surveillance.