Bashor et al. (1) very nicely demonstrate the importance of specific mutations in the switching of severe acquired respiratory syndrome coronavirus 2 (SARS-CoV-2) between in vivo and in vitro. They also identify 14 mutations that emerge when SARS-CoV-2 infects the animal species they study. However, their data analysis regarding low-frequency variants needs significant modification. Correct data analysis of low-frequency intrahost variants is also crucial for the calculation of transmission bottlenecks and associated disease mechanisms (2, 3). The learned authors of ref. 1 claim that viral titer does not matter for how much variant richness is found. As evidence, they quote selected examples where low titer samples and high titer samples have similar numbers of variants (for instance, dogs vs. specific cats and hamster 1 vs. hamster 3). This claim is not tenable. In Fig. 1/4 (4), as high-depth samples are down sampled, the number of intrahost-single nucleotide variants (iSNVs) detected first increases and after reaching a peak, decreases. The samples quoted by Bashor et al. (1) could just be on different sides of the hump, or some samples could be on lower peaks and other samples on the sides of other curves, thus giving an illusion of invariance of the iSNV count with change in titer.
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