Introduction
The COVID-19 outbreak caused by SARS-CoV-2 has led to major socio-economic disruption worldwide and the pandemic has yet to secede after more than 18 months. As of November 2021, SARS-CoV-2 has infected over 251 million people and caused over 5 million deaths worldwide. The discovery of aggressive variants of the virus has shown that monitoring for variants associated with changes to infection outcome and transmission of SARS-CoV-2 is critical to ensure the success of vaccination programs and establish robust public health responses. A sample identified positive for COVID-19 using an RT-PCR assay, then that sample can be tested to determine if it has a SARS-CoV-2 Variant of Concern (VOC). Next Generation Sequencing (NGS) is ideal for this as its high resolution enables scientists to identify all variants, known and unknown, providing insights to SARS-CoV-2 viral evolution.
Methods
Our laboratory is one of the largest COVID testing laboratories set up at apex largest outbreak of SARS-CoV-2 patients California state had seen since the beginning of the pandemic. The laboratory was set up in a total of 8 weeks which included reagent supply, staff, and operate a high-throughput, large scale SARS-CoV-2 testing facility which is capable of processing 150 thousand samples per day. To date, our laboratory has tested over 6 million patient samples for COVID-19 since its inception in November 2020 with a total positivity rate of 8.07% at this reporting. Due to the labs’ ability to process SARS-CoV-2 patient samples at such a high-throughput with a turnaround time averaging 12.2 hours per sample, we quickly utilized our ability to rapidly mobilize the lab’s skilled scientists to efficiently implement NGS at the lab. We exercised the collaborative efforts of the lab team along with experts from multiple other divisions including R&D, Genomics, and Business to build, test, and release, for research use only, a proprietary NGS sequencing kit that utilizes robust NGS reagents in conjunction with a community designed Artic Primer set. Discussion in the lab about implementing NGS took place in February 2021 and by March 2021, we were fully operational and had begun to sequence these positive samples for epidemiological purposes. A streamlined process was established within four weeks using robotic automation as well as training an inexperienced staff running 4 shifts for a 24/7 lab operation. The laboratory utilizes two Illumina platforms, the NovaSeq and MiSeq systems to process high sample throughput and high priority samples. The use of our proprietary NGS kit allows for 1536 unique barcodes so that a high throughput of samples can be run on one flowcell, and this kit design allowed for our lab to utilize a full Illumina S4 flowcell with approximately 6144 samples at a time.
Results
At this reporting, more than 115,000 samples have been sequenced using the NGS kit to determine the populations of VOCs rampant in California. This is broken up into two workflows: routine high throughput processing on the NovaSeq with a turnaround time of 7 days and STAT sample manual processing on the MiSeq with a turnaround time of 48 hours. The lab can process up to 24 STAT samples daily and an average of 5000 routine patient samples per week. SARS-CoV-2 in patient samples is measured by qPCR; where having a cycle threshold (Ct) cutoff value determines the presence and amount of virus detected in a sample. Of these samples approximately 35% (∼39,200) have been weakly detected with a Ct value greater than 33. Samples with high Ct values, which are indicative of lower viral titer traditionally do not meet the requirements for sequencing set forth in other laboratories. Lower viral titer leads to lower nucleic acid concentration which causes under clustering leading to total flowcell failure and more importantly, missed patient results.
Conclusion
Due to our high throughput automated NGS workflow plus the robust NGS reagents in conjunction with a community designed Artic Primer set, our laboratory was able to successfully sequence 40% of these high Ct value sample which would have been rejected elsewhere. Due to our laboratory’s unique sequencing methods, the government is able to closely monitor and track emerging VOCs within the state. We have traced the increase of pathogenic variants such as Delta as it first appeared in California, to now circulating in the population at 85% of all VOCs detected. This epidemiological information is a crucial tool in fighting the COVID-19 pandemic, providing relevant data to authorities and researchers to understand SARS-CoV-2 infection and transmission.