Optimization of Illumina AmpliSeq protocol for SARS-CoV-2 and detection of circulating variants in Goiás State, Brazil from November 2020 to July 2021

ABSTRACT

The SARS-CoV-2 pandemic has demonstrated the need for genomic epidemiology surveillance. To date, various methodologies have been applied, including metagenomic approaches and amplicon-based sequencing associated with high-throughput sequencing platforms. We adapted some details in amplicon-based sequencing using a SARS-CoV-2 community panel (Illumina AmpliSeq), with additional modifications for balanced and high-quality sequencing using the MiSeq platform. The modified protocol was used to detect circulating SARS-CoV-2 variants in Goiás state, Brazil. Initially, RNA samples were obtained from swab samples from 15 patients from the state of Goiás, Brazil, in November/2020 and February/2021 to validate protocol steps. The libraries were prepared following AmpliSeq for Illumina workflow with modifications;subsequently, we analyzed 305 positive samples collected from the state of Goiás from December 2020 to July 2021. For protocol improvement, we removed the need to treat samples with DNAse and demonstrated the importance of quantification by qPCR before and after library dilution. No fragmentation pattern was observed in the samples analyzed with Bioanalyzer. The libraries returned sequencing results that were used for genome assembly and variant detection. We were able to assemble SARS-CoV-2 genomes from 318 samples, which were used to identify 13 variants of coronavirus circulating in Goiás throughout those months. Variants of concern, such as Alpha (B.1.1.7), Gamma (P.1) and Delta (B.1.617.2) were detected;the latter was detected at first in Goiás in April 2021. The modifications in the workflow we developed were successfully applied to detect SARS-CoV-2 variants, resulting in high coverage genome assembly, and they can be used to increase the number of genome sequences and aid in epidemiological surveillance in Brazil.