Figure 4

Sequencing results. Sequencing results for libraries prepared in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%) using the custom bisulfite PCR multiplex assay. (A) The proportions of each pool across 54 libraries, determined as a total amount of each library. Y-axis: Percentage of total library = Total number of reads for a pool ÷ Total number of reads for the library. Although pool 7 was observed to dominate all libraries, the proportion of each pool across 54 samples was maintained at consistent levels. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (B) The average standard deviation in 8-plex pool proportions observed across all libraries. Average pool standard deviation = (The sum of all standard deviations for a single pool ÷ The total number of entries, that is, the mean value). Across 13 FFPE samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries. (C) The proportion of each amplicon in each of the libraries, calculated as a percentage of its original 8-plex pool. Percentage pool proportion = Total number of reads for an amplicon ÷ Total number of reads for its pool. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (D) Histogram showing the distribution of the average standard deviations for all 57 amplicons in the assay. Across 13 FFPE samples amplified in triplicate, the proportion of each of the 56 amplicons was maintained at consistent levels. Average standard deviation = The sum of all standard deviation values for a single amplicon (as a percentage of its original 8-plex pool as outlined in C) ÷ The total number of entries.