Sabine Hazan, MD, Andreas Papoutsis, PhD, Brad Barrows, DO; ProgenaBiome, Ventura, CA
Introduction: In this study we sought to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by next generation sequencing (NGS) from fecal collections. We also aimed to characterize unique SARS-CoV-2 genome variations among the patients to capture viral evolution that may be occurring in the population Methods: Study participants (n = 10) underwent testing for SARS-CoV-2 from fecal samples by NGS. Following fecal collection,RNA was extracted, reverse transcribed, enriched, and sequenced on Illumina’s NextSeq 550 System. Sequences were mapped to the SARS-CoV-2 Wuhan-Hu-1 (MN90847.3) genome utilizing One Codex’s SARS-CoV-2 analysis pipeline. SARS-CoV-2 positive samples were further analyzed for mutational variants that differed from the reference genome. 8 study participants had their nasopharyngeal swabs tested for SARS-CoV-2 by real-time polymerase chain reaction (rt-PCR). Results: Of the 10 study participants, seven tested positive for SARS-CoV-2 by rt-PCR, one asymptomatic individual tested negative, and two did not undergo rt-PCR testing (Fig. 1). The concordance of SARS-CoV-2 detection by NGS from stools among positive patients by rt-PCR nasopharyngeal analysis was 85.7% (6/7). Patient 8, who did not undergo nasopharyngeal analysis, was positive by NGS. Asymptomatic patients 9 and 14, were negative by NGS. All positive samples analyzed by NGS achieved 100% genome coverage of SARS-CoV-2 except for patient 10’s sample, which had 93% coverage (Fig. 2). SARS-CoV-2 variants at positions 241 (C → T) and 3037 (A → C) were found across all positive patients, and variants at positions 23403 (A → G) and 25563 (G → T) were each found among six of the seven patients (Fig 3). Patient 1 harbored the most unique SARS-CoV-2 genome, with six variants not found in any of the other individuals. Figure 3 highlights the 4 distinct SARS-CoV-2 genomes among the 7 patients, with a total of 23 different mutations. Interestingly, 6 of the patients all harbored a mutation at position 23403, in the spike glycoprotein region, involved in ACE2 receptor binding. Discussion: Although previous studies have identified SARS-CoV-2 in fecal collections by rt-PCR, this is the first study to our knowledge, to report whole genome sequencing (WGS) of SARS-CoV-2 from stool samples. The ability of NGS to identify unique genomic differences among fecal collections, may provide an alternative source for COVID-19 identification, and tracking of its evolutionary progression through the population.
Comparison of patients’ SARS-CoV-2 rt-PCR test results from nasopharyngeal swab samples and SARS-CoV-2 NGS results from fecal samples.