Quantifying sequencing error and effective sequencing depth of liquid biopsy NGS with unique molecular identifier error correction

Two widely used Illumina library preparation kits were benchmarked for next-generation sequencing of cell free tumor DNA: One library type without unique molecular identifier (UMI), but in technical duplicates and one library type with UMIs. Reference cfDNA was used with 0% mutation as well as with a mutation in PIK3CA of 0.13% tumor allele frequency. PIK3CA was enriched using custom baits and sequenced to 50,000X average on-target coverage on an Illumina instrument using 2x150 bp paired end reads. Illumina's UMI Error Correction Local App was used for aligning and collapsing UMI sequences. Signal noise defined by false-positives in UMI- and non-UMI libraries was compared, and effective sequencing depth loss due to the bioinformatic processing was quantified to allow for estimating the required sequencing depth.