Severe acute respiratory syndrome coronavirus 2 spike ARTIC amplicon 76 dropout in relation to primers, viral load, and variants
Authors: Haggag Maha G. , Tagliamonte Massimiliano S. , Cash Melanie N. , Mavian Carla N. 
Published: December 2022

Background/aim
In January 2020, the ARTIC Network designed 98 pairs of primers divided into two pools for targeting amplification of the severe acute respiratory syndrome coronavirus 2 genome by multiplex PCR. However, in using this protocol, several users, including our team at Emerging Pathogens Institute at University of Florida, noticed a systematic dropout of reads covering amplicon 18, which overlaps the gene for nonstructural protein 3 (nsp3) in ORF1a, and amplicon 76, targeting the spike (S) gene. The aim of this work was to verify the design of primers for the dropout region of amplicon 76 in the V3 ARTIC protocol covering the severe acute respiratory syndrome coronavirus 2 spike gene, to evaluate correlation of dropout to viral load and determine if there is a correlation between dropouts and viral load and viral lineages and as well as to further explore potential mutations in the primer regions.

Materials and methods
A total of 544 samples presenting dropout at amplicon 76 from the V3 ARTIC primer set were collected over time from December 2020 to December 2021 in Alachua County, Florida. RNA was extracted, followed by cDNA synthesis and PCR amplification, which targeted the dropout region within the spike. PCR amplification was performed with forward and reverse primers designed in-house. Sequencing was performed to detect potential mutations at primer sites. Viral load was performed for 96 samples showing a dropout of the spike genome at amplicon 76. The results were compared and correlated to lineage/variant of concern.

Results
A total of 544 dropout samples were amplified with the primers designed in-house. Overall, 381 (70%) of these samples showed bands visible by gel electrophoresis. This result, along with Sanger sequencing, was enough to verify the efficacy of our designed primers in amplifying the dropout region. Sequencing revealed that primer regions were conserved; no mutations were observed at the site corresponding to the Illumina primers for amplicon 76. The mean Ct value of dropouts was 32.53, and the mean Ct value of nondropouts was 26.11. Welch test on Ct values of dropouts versus nondropout showed significant difference (P<10−6). The 96 dropout samples tested for viral load included 17 different lineages, with delta lineage (B.1.617.2-like) being the most common (21.8%), followed by B.1.2 (10.4%), alpha (B.1.1.7-like) (9.4%), B.1.234 (9.4%), iota (B.1.526-like) (5.2%), and gamma (P.1-like) (4.2%). Unexpectedly, when determining the effect the lineage on the chance of a sample being a dropout, the delta lineage had an odds ratio of less than 1, implying that a sequence belonging to delta lineage was less likely to produce a dropout.

Conclusions
Our study showed that dropouts are not due to mutation at the primer sites. Viral loads likely affect the odds of a sample presenting a dropout of amplicon 76 but was not the only cause of dropout. Several dropout samples could not be classified because of undefined genomic segments, which may have skewed the resulting odds of lineage on sequencing. It is possible, as previously suggested, that dropouts may be due to interaction and dimer formation between primers of the multiplexed PCR reaction.