Fig. 2: Refined sequencing methods and robust variant calling methodology enables confident analysis of variants from autopsy tissues.
A Schematization of enhanced sequencing methods. B Two independent libraries from four samples with high viral depth of coverage (>3,000x mean coverage) were downsampled to a range of mean coverage depths (90x to 1,250x), and variant profiles were identified in each condition, then compared to those detected in the highest coverage condition (>3,000x mean coverage). For each library from all four samples, at each coverage depth condition, precision and recall (top; points represent the mean, while error bars represent standard deviation), the number of variants identified (middle; boxes delineate quartiles, whiskers delineate range excluding outliers), and the frequency distribution of variants (bottom; points represent variants across all samples within a condition) were compared to the highest depth of coverage condition. Green represents conditions >500x mean depth of coverage, the threshold selected for high resolution genomic analyses. C For 12 samples, the same libraries were sequenced with and without hybrid capture enrichment, and were then downsampled to the same number of raw reads; mean depth of viral coverage was calculated and plotted for each sample. The order of magnitude “OM” of enrichment is annotated across the two-dimensional space. D Variants were identified for all samples in the sample set with at least 500x mean depth of coverage, including those sequenced with hybrid capture enrichment (purple) and without (blue). Frequency of variants identified in two independent libraries were compared (top), demonstrating high correlation (R2 = 0.998). Number of variants identified (middle) and frequency of variants identified (bottom) was compared across samples, each showing poor correlation with viral load (R2 = 0.229 and R2 = 0.167, respectively).
