The effects of input DNA quality and quantity on variant detection background distributions. A) The figure shows variant calling background for the indicated mass amounts of intact cell line or comprised quality, FFPE sample DNA. The standard deviation of the heterozygous variants increases only slightly with reduced DNA input quantities with intact DNA (left panel), but increases dramatically when the quantity of lower quality, FFPE DNA is reduced (right panel). The 99th percentile of the background percent variant (red and blue lines) is more consistent for cell line DNA than for FFPE DNA with the rise in the background being largely driven by the GC > AT transitions. B) The median 99th percentile of the background for all possible substitutions (with G > A and C > T collapsed into GC > AT) from TAS analysis using 250 ng DNA from 8 FFPE samples using the 1052-amplicon panel (x-axis) compared to after target enrichment using an independent 35-amplicon panel (y-axis). As expected, GC > AT transitions contribute higher background than other possible substitutions.