Comparison of read alignments with and without primer bases. The top panel shows alignments of reads without primer bases and the bottom panel shows alignments of reads with primer bases. For reads without the primer bases, the G- > A variant near the end of the read causes the read aligner to exclude a portion of the amplicon insert from the alignment, while the reads without the variant are aligned completely. Because the variant base is not part of the alignment, it is impossible to call the variant. In contrast, for reads with the primer bases (bottom panel), the complete amplicon insert region is aligned, thereby allowing the G- > A variant to be called. In the bottom panel, the primer bases have been soft-clipped by us after obtaining the alignments with the complete reads; i.e., the read aligner did not soft-clip the alignments in the bottom panel.