High-throughput next-generation DNA sequencing of SARS-CoV-2 enabled by the Echo 525 Liquid Handler

“The Echo 525 Liquid Handler is unmatched when it comes to preparing miniaturized amplification reactions in a scalable manner. With the Echo 525, we can assemble reactions in minutes that would otherwise take hours or days, all directly from primers at stock concentrations. This capability of preparation of PCR reactions without tips at scale, is critical in eliminating throughput bottlenecks for our SARS-CoV-2 assay.” - Dr. Jeremy Ellis, Chief Scientific Officer at Fry Laboratories, LLC

Introduction

SARS-CoV-2, a novel coronavirus that causes coronavirus disease 2019 (COVID-19), originated in Wuhan, China in late 2019 and has triggered a global pandemic [1].

Fry Laboratories, LLC, a leader in targeted next-generation DNA sequencing (NGS) assays [2], responded by developing and expanding their current assays to target specific amplicon sequences of the SARS-CoV-2 virus, in 1,152 samples in a single sequencer run, simultaneously. The assay uses SARS-CoV-2 N1, N2, and N3 viral targets that are run in parallel with the human RNase P gene (RP) control. Unlike RT-qPCR detection assays that rely on probe complementarity and the detection of probe degradation, this assay sequences the PCR-generated amplicons. False negative results due to mismatches between the probe and the viral targets do not impact an NGS-based assay, as the entirety of the target is sequenced, base-per-base. False positive signals may occur in RT-qPCR assays due to the unintended probe degradation or amplification of unintended PCR targets. Sequencing-based tests do not incur this source of error. Furthermore, NGS eliminates the most common sources of false positive and false negative results. In sum, while the assay is developed for SARS-CoV-2 , the robustness and performance characteristics of this method is compatible with viral surveillance, prevalence, and mutation characterization research.

Scalability, reproducibility, and precision are characteristics required for high-throughput molecular assays. Using focused sound waves, the Echo 525 Liquid Handler dispenses liquids with 25 nL precision into PCR destination plates. This capability allows laboratories to assemble PCR reactions, on demand, directly from concentrated primer stocks. Dispensing highly concentrated oligos allows for a corresponding reduction in reaction volume (from 20 µL reactions to 10 µL reactions), while keeping input template volume (5 µL) identical (Table 1). Our preliminary data demonstrates successful miniaturization of our reaction mixes using the Echo, with an extremely sensitive LOD at approximately 60 viral copies per mL in saliva samples. This is well within clinically relevant ranges given the 10⁴ and 10⁸ copies per mL found in infected individuals [3].

“Implementation of the Echo 525 in our workflow has eliminated the consumption of 2,312 tips per sequencing run. PCR reaction miniaturization using the Echo allowed us to consume 50% less RT-PCR master mix with little change to our workflow. Simply, the Echo allows us to print PCR reactions – on demand – with the ease of a laser printer.” - Dr. Jeremy Ellis, Chief Scientific Officer at Fry Laboratories, LLC

Workflow

200 µL of saliva collected without fixative or holding media was extracted using the RNAdvance RNA Viral Extraction Kit (Beckman Coulter Life Sciences), yielding 40 µl of purified nucleic acid. This total RNA mixture containing putative SARS-CoV-2 RNA and human RNase P target, is reverse transcribed and the specific targets (N1, N2, N3, and RP) are PCR amplified using the GeneExpression Master Mix (IDT) using indexed and adapted fusion primers [4].

Figure 1 – Sample and workflow overview for the SARS-CoV-2 NGS Assay

Reaction miniaturization with the Echo 525 liquid handler.

The Echo 525 can dispense primers directly from concentrated oligo stocks (50 – 100 nM) in 25 nL increments, allowing for a reaction to primarily consist of the input template (5 µL) and the PCR master mix (5 µL), resulting in no reduction in the volume of template per reaction. As such, the Echo allows for efficient miniaturization of the amplification reactions, reducing the master mix consumption by half.

Table 1 – PCR Reaction Mixture Comparison

Testing the assay’s full throughput capacity of 1,152 samples requires the assembly of 2,304 PCR reactions (a set of reactions for the SARS-CoV-2 targets and an independent set for the RP control targets). These reactions consume 2,312 tips at minimum when distributing pooled primers. The same 2,304 PCR reactions may be assembled utilizing no tips with the Echo’s acoustic, tip-less liquid dispense technology. Further, the ability to directly prepare PCR reactions from individual stock concentration oligos reduces mixing and formulation errors during primer pooling and enables exhausted primers to be replaced as needed. This feature, in addition to the reduction in master mix consumption directly addresses two of the primary fixed-assay costs.

Data

Preliminary LOD (Limit of Detection) comparison between the Echo 525 liquid handler and a manual method.

On average 5,738 reads per sample index were obtained. As expected, as the input virus copy number increased the percent of reads for the viral targets (N1, N2, and N3) increased with a corresponding decrease in the RP control target (Table 2). Multiple factors, including the number of reads, the proportional distribution, and other sequence specific features, contribute to the final positive, negative, or retest result call as determined by the analysis algorithm.

Table 2 – Average Read Depth and Target Proportion

The LOD, defined as a 95% detection rate as determined by Probit Analysis, for the SARS-CoV-2 assay using the manual method [5] was determined and compared to preliminary LOD generated using the Echo 525 liquid handler [6]. The observed LOD for the standard 20 µL and the Echo 10 µL reactions exhibited:

• Approximately 47.0 SARS-CoV-2 copies per mL of saliva using the standard 20 µL reaction in a 96-well PCR plate, with 5 µL RNA template (n = 71 reactions)
• Approximately 69.5 SARS-CoV-2 detection per mL of saliva using an Echo assembled 10 µL reaction in a 96-well PCR plate, with 5 µL RNA template (n = 12 reactions)

Figure 2 – SARS-CoV-2 LOD Probit Analysis Virus Spiked Controls

The LOD was determined by Probit Analysis of results, utilizing saliva spiked with SARS-CoV-2 at 0 copies/mL, 2.5 copies/mL, 5 copies/mL, 25 copies/mL, and 100 copies/mL (Figure 2). The observed LOD, 47.0 copies/mL vs 69.5 copies/mL, exhibited comparable performance.

Indistinguishable performance was expected as the volume of the template was held constant between the protocols. Further, 50 copies/mL would result in the processing of, on average, 10 virus particles by the RNA extraction (200 µL saliva). This would result in an effective viral genome concentration of 0.25 copies per µL (10 copies in 40 µL elution). The assay uses an input of 1.25 genomes (5 µL of template RNA) that approaches the theoretical stoichiometric limits. Given the physical limits of the assay, it is expected that additional replicates of the Echo protocol will perform nearly identically to the standard EUA submitted protocol. In sum, the derived LOD is well below the concentration of SARS-CoV-2 as detected in infected individuals [3].

Additional replicates will be used to establish the final sensitivity, specific, positive and negative predictive values of the assay. We also plan on evaluating the Echo for downstream library pooling and simultaneous quality control checks, and we predict that these uses will further demonstrate the value of the Echo 525 in an NGS-based workflow. Lastly, the print-on-demand capability of PCR reactions will be critical as our assay evolves to include emerging SARS-CoV-2 strain typing capability as the pandemic continues into 2021.

Summary

The SARS-CoV-2 pandemic has resulted in significant morbidity and mortality both within the United States and internationally, with concomitant substantial interruption of commercial activity. Widespread SARS-CoV-2 screening is a cornerstone to re-establish domestic economic production and global health. Fry Laboratories has developed and validated a high-throughput assay using the Illumina MiSeq DNA sequencing platform. This method allows each Illumina MiSeq to process 1,152 samples with a 4 hour on sequencer run time. This capability is possible due to an innovative approach that simultaneously reverse-transcribes, amplifies, barcodes, and adapts the SARS-CoV-2 N1, N2, N3, and the human RNAse P targets for rapid next-generation DNA sequencing. Assembly of the complex and critical PCR reactions for this assay is critical for performance and the cost-effectiveness of the assay. The Echo 525 Liquid Handler supports scalability, efficiency and cost effectiveness, providing significant savings that reduce bottlenecks that can influence adoption of this protocol. The capability showcased by the Echo can translate to any public and private laboratory that has similar amplicon-based assays.

Highlights

Fry Laboratories adopted the Echo 525 Liquid Handler to assist in establishing scalable complex multiplex PCR reaction assembly. Fry Laboratories compared their existing standard SARS-CoV-2 NGS detection assay workflow to a slightly modified protocol utilizing the miniaturization and any-well-to-any-well dispense capabilities of the Echo 525 Liquid Handler. In addition to the immediate time and tip savings, the laboratory was able to assemble reactions directly from concentrated primer stocks thereby reducing the overall reaction volume and reagent consumption. These benefits were realized with no significant impact on assay performance, and were established within 2 weeks of the Echo installation.

Overall, the Echo has resulted in several impactful and significant improvements or capabilities:

• Dispense High Concentration Stock Oligos: Dispensing 25 nL of highly concentrated oligos directly from the stocks (effectively 0 volume) enables a more efficient template to total volume ratio.
• Reduced Reaction Assembly Time: > 60% reduction (45 minutes for a full run from 2 hours with pre-pooled stocks). Additionally, if the reactions were assembled directly from oligos stocks, mimicking how the Echo works, we estimate it would take 40-50 manual labor hours.
• Decreased Reagent Consumption: 50% reduction of master mix consumption (5 µL from 10 µL)
• Immediate Tip Consumption Savings: More than 4,500 tips per run are saved.
• Dispense Complex Mixes with Ease: Using the Echo “picklist” format, the lab was able to set up and perform preliminary tests with the Echo within two days after installation and set up a print-on-demand workflow.
• Sequencing Library Pooling: Nanoliter-level pooling of libraries reduces sample-to-sample variability by improving precision, reducing consumption of rare amplicons, and enabling more precise quality control and sample archiving.

Acknowledgement

We would like to thank the Fry Laboratories, LLC research team for highlighting their work using the Echo.

References

1. Zhu, N. et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727-33. https://doi.org/10.1056/NEJMoa2001017
2. Ellis, JE, et al. Rapid Infectious disease identification by next-generation DNA sequencing. J Microbiol Methods 2017: 138:12-9. https://doi.org/10.1016j.mimet.2016.09.012
   
3. Zhu J, et al. Viral dynamics of SARS-CoV-2 in saliva from infected patients. J Infect. 2020: Sep; 81(3): e48-e50. https://doi.org/10.1016/j.ijinf.2020.06.059
4. National Center for Immunization and Respiratory Diseases (NCIRD), Division of Viral Diseases. CDC’s Diagnostic Test for COVID-19 Only and Supplies. Updated 2020: Dec 9. https://www.cdc.gov/coronavirus/2019-ncov/lab/virus-requests.html
5. Ellis, JE, et al. Fry Laboratories SARS-CoV-2 NGS Assay. 2020: Nov; EUA203034.
6. CLSI EP17-A2. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA, 2012.

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