Leveraging enhanced conversion efficiency of the KAPA EvoPrep Boost workflow for urine ctDNA detection

Bladder cancer is the 9th most common cancer type globally, and early detection is often associated with a better prognosis. While tissue biopsy is the gold standard, its invasiveness limits disease detection and monitoring. Liquid biopsy is a less-invasive alternative, typically using plasma to monitor for circulating tumor DNA (ctDNA). In recent years, urine has emerged as a potentially superior source of ctDNA in bladder cancer studies, as it comes in direct contact with the tumor tissue 1 . However, high concentrations of DNAases in urine result in shorter cell free DNA (cfDNA) fragments and a lower concentration of cfDNA in the sample. Accurate analysis therefore relies on a sensitive and efficient sample preparation workflow. The KAPA EvoPrep Boost workflow employs an engineered ligase and an optimized amplification system, which improve library conversion efficiency and uniformity. The enhanced library preparation performance makes it a promising tool for analyzing poor quality and low input cfDNA obtained from urine. Urine cfDNA (ucfDNA) was isolated from healthy donors and archived bladder cancer samples and characterized. UcfDNA was converted using KAPA EvoPrep Boost and another commercially available library preparation kit for comparison, followed by target enrichment using the KAPA HyperCap Oncology Panel, which is a 214 Kb (capture) target enrichment panel covering the coding region of 13 genes involved in somatic oncology in addition to hotspot variants across 69 genes. The libraries were sequenced on an Illumina NovaSeq 6000 System and analyzed with a Roche KAPA Somatic DNA RUO Pipeline. Population databases of known single nucleotide polymorphisms were used to filter out putative germline mutations given matched normals were not available for the bladder cancer samples.Using Seraseq ctDNA Complete Reference Material, assay sensitivity in detecting variants from common oncogenic regions was established at 1ng cfDNA input. Compared to the alternative library preparation kit, KAPA EvoPrep Boost achieved higher coverage uniformity and lower error rates when tested with low input ucfDNA samples from healthy donors. Furthermore, using the KAPA EvoPrep Boost workflow coupled with the KAPA HyperCap Oncology Panel, variants in genes that are often mutated in bladder cancer development were detected in the cell-free DNA shed into the urine by the tumor. In summary, we describe a complete workflow for the analysis of low input and potentially poorer quality cfDNA isolated from urine. The improved conversion efficiency and genome equivalent recovery rates achieved by using the KAPA EvoPrep Boost Kit will be a useful research tool for bladder cancer and potentially other urinary tract cancers.KAPA products are For Research Use Only. Not for use in diagnostic procedures. 1.Ou Z, et al . Detection of bladder cancer using urinary cell-free DNA and cellular DNA. Clin Transl Med. 2020; 9(1):4.