Orthogonal Confirmation of SARS-CoV-2 RT-PCR Results with NGS

The PerkinElmer® New Coronavirus Nucleic Acid Detection kit has been identified as having the highest sensitivity of the commercially available COVID-19 diagnostic kits1.  The purpose of this study is to use Next Generation Sequencing (NGS) to confirm positive COVID-19 samples with a range of Ct values to verify the presence of the SARS-CoV-2 genome.


A second aliquot of extracted genomic material was taken from COVID-positive nasopharyngeal samples as determined using the PerkinElmer® New Coronavirus Nucleic Acid Detection kit and used as template for NGS.

Eight microliters of undiluted extracted nucleic acid sample were reverse transcribed and amplified using publicly available SARS-CoV-2 genome-specific ARTIC v3 primer set along with all reagents needed for reverse transcription, amplicon PCR and NGS library preparation. Equal volumes of amplicon pools 1 and 2 were combined and purified, then 35 μL was enzymatically fragmented and prepared for NGS. Following quantification, the samples were pooled in equivalent quantities per standard library normalization protocol.

NGS fastq files were trimmed of adapters via Trimmomatic and aligned to the SARS-CoV-2 Wuhan-hu-1 reference genome (NC_045512.2) via Bowtie2. Intermediate alignment file formatting was performed with Samtools. Primer sequences were removed from aligned reads using iVar’s trim command, and consensus sequences were then generated using iVar’s consensus command. Duplicate reads were removed from the bam files before visualization in IGV version 2.8.10 using the MN908947.3 reference genome. The removal of duplicate reads enabled clearer visualization and did not impact the results.

Table 1: Species used in alignment


From a representative NGS run, ninety-eight regions spanning the entire SARS-CoV-2 genome were analyzed to identify target areas with more than 10X read depth. This was compared against RT-PCR Ct values.

Table 2: Orthogonal confirmation of RT-PCR data using NGS

A total of one hundred and forty-nine samples spanning Ct values from 10 to 42 obtained using the PerkinElmer® New Coronavirus Nucleic Acid Detection kit, including 38 samples with a Ct value more than 37, were analyzed by mapping to the SARS-CoV-2, human and other reference genomes. Samples have been named by sample number based on sorted increasing average Ct, followed by the sample N gene Ct, and ORF1ab Ct, all separated by dashes. If only one target gene signaled, the absent gene’s Ct value is written as “NULL.” Rows in the heat map in Figures 1 and 2 represent species and columns represent samples. The heatmap is scaled linearly from 0 to 100% of sample reads aligning to each species.

No sample has 0% alignment to SARS-CoV-2 and no sample aligns better to any species besides SARS-CoV-2 or human. This confirms the presence of SARS-CoV-2 in samples associated with a wide range of Ct values, including high Ct values, generated using the PerkinElmer® New Coronavirus Nucleic Acid Detection kit.

Figure 1: Cross-reactivity species alignment of 149 nasopharyngeal swab samples
Figure 2: Cross-reactivity species alignment of the subset of 38 samples analyzed with Ct values higher than 37 for one or both SARS-CoV-2 qPCR targets.


This data confirms presence of SARS-CoV-2 in samples associated with a wide range of Ct values including high Ct values generated using the PerkinElmer® New Coronavirus Nucleic Acid Detection kit.


    1. accessed 10 Feb 2021
    2. accessed 10 Feb 2021.

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