Advantages of Single-Stranded DNA Over Double-Stranded DNA Library Preparation for Capturing Cell-Free Tumor DNA in Plasma

PURPOSE: Single-stranded DNA (ssDNA) library has shown to enrich shorter and more degraded DNA fragments than double-stranded DNA (dsDNA) library. In this study, we evaluated whether ssDNA libraries capture more circulating tumor DNAs (ctDNAs) in plasma cell-free DNA (cfDNA). MATERIALS AND METHODS: We prepared dsDNA, ssDNA and pure-ssDNA (capture the pre-existing ssDNA) libraries using ten plasma cfDNA samples. After low-pass whole genome sequencing, we calculated duplicate rate to estimate library complexity and compared the library insert sizes between different library methods. Finally, we estimated ctDNA content and plasma genomic abnormality (PGA) score, an indicator of ctDNA burden. RESULTS: 27 libraries were prepared and sequenced from the ten cfDNA samples. Duplicate rate in the ssDNA and pure-ssDNA libraries was significantly lower than dsDNA libraries (p< 0.001 and p< 0.01, respectively). ctDNA content and PGA scores were consistently higher in ssDNA and pure-ssDNA libraries than in matched dsDNA libraries (p< 0.005). The higher ctDNA content in ssDNA libraries was associated with smaller library insert size. CONCLUSIONS: ssDNA libraries preserve more diversity and capture more ctDNA than dsDNA libraries. The ssDNA library method is preferred when performing genomic analysis of ctDNA.