Comparison of different sequencing techniques with multiplex real-time PCR for detection to identify SARS-CoV-2 variants of concern

ABSTRACT

As different SARS-CoV-2 variants emerge and with the continuous evolution of sub-lineages of the delta and other variants, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, as many resource-poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using the Illumina generated <15% ambiguous bases. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. Sub-consensus single nucleotide variations (SNV) were highly correlated between both platforms (R2=0.79) while indels showed a weaker correlation (R2=0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number of reads, generated less ambiguous bases and was significantly cheaper than Illumina sequencing technology.