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In addition to known positives, we also determined a set of negative positions. The total read depth (DP), reference allele depth (RDP) and alternative allele depth (ADP) were counted with the samtools [24] (v1.9) mpileup function at each position. Positions with DP < 50 in one library were removed. DP and RDP of the two libraries for each cell line were summed together and the reference allele frequency (RF), alternative allele frequency (AF) recalculated. We then applied filters by merged DP ≥ 125 and recalculated RF ≥ 99%. A simple averaged RF of the ten cell lines was calculated and filtered by average RF ≥ 99.5%. We only considered negative positions inside the CTR (which is a subset of the intersection of the coding target regions for the WES1–3) as used for the Class 1 positives. We did this procedure for both genome versions hg19 and hg38. We then lifted over the hg19 version to hg38 (noted “hg19ToHg38”) and hg38 version to hg19 (noted “hg38ToHg19”) with R package rtracklayer [100] (v1.42.0). For INDEL calls with the simple samtools mpileup, the adjacent positions from both ends of the INDEL calls were removed. To further remove possible variants, we employed a similar route to call negative positions through WES1–3 data of pooled Sample A. The DP and RDP of all libraries of each of WES1–3 were summed together. Any positions with total DP ≥ 500, ADP > 2, and AF ≥ 0.002 were removed. The adjacent positions from both ends of the INDEL calls were also removed. Finally, we took the intersection between hg19 and hg38ToHg19, or between hg38 and hg19ToHg38 as known negatives for the pooled Sample A in this study. This stringent process led to 10,229,649 negative positions in hg19 and 10,208,086 negative positions in hg38.

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