Abstract
In December 2019, a novel coronavirus emerged in Wuhan China, later to be classified as SARS-CoV-2 and commonly referred to as COVID-19. This outbreak eventually spread to a global pandemic that has affected over 132 million people worldwide. Our goal was to develop optimized methods to sequence the whole SARS-CoV-2 genome so we could comprehensively track and monitor various outbreaks, variants, and intra-patient variation throughout the current SARS-CoV-2 outbreak in the Nebraska region. Here, we streamlined a sequencing pipeline by optimizing several factors at each step of the process including initial sample quality, RNA isolation method, and sequencing platforms which all contribute to quality sequencing results. We experimentally determined that the Maxwell automated RNA extraction method was the preferred method for vRNA isolation. We experimentally determined the minimal sample quantity of vRNA was 104 copy numbers to obtain full genome coverage. We also determined the isolation threshold for SARS-CoV-2 from clinical samples is 104 copy numbers. Based on the diagnostic CN values, which are a proprietary value given by the diagnostic machine, given from the CHI Health Creighton University Clinical Laboratory, samples with a CN under ten were determined to have the highest likelihood of complete sequencing coverage. By comparing two sequencing platforms, MinION and Illumina, we determined that the Illumina platform was the preferred method based on error rates, sequencing read depth, and multiplexing compatibility. Using our pipeline, we calculated the mutation rate of SARS-CoV-2 in twelve individuals who were infected with SARS-CoV-2 and had four or more positive specimens ranging from 12-52 days. Within our patient data we identified eight common SNPs, five of which are commonly reported variants located at genomic positions 1059, 3037, 14408, 23403, and 25563. Based on our regression analysis, the viral rate of mutation in patients decreases after about 21 days. This suggests that prolonged infection does not result in a steady rate of viral evolution within individuals.