Abstract 3536: Advantages of ssDNA over dsDNA library preparation for capturing cell-free tumor DNA in plasma
Authors: Zhu Jing , Huang Jingyong , Zhang Peng , Kohli Manish , Huang Chiang-Ching , Wang Liang 
Published: August 2019

Single-stranded DNA (ssDNA) sequencing library method has shown to enrich shorter and degraded DNA fragments than double-stranded DNA (dsDNA) library. The goal of this study is to determine whether ssDNA libraries enriches more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA). We used 2ng cfDNA to prepare dsDNA libraries and ssDNA libraries. We also prepare pure-ssDNA libraries by skipping the DNA denaturation step to capture the pre-existing ssDNA only. To calculate ctDNA content, we mapped fastq files to human genome and binned the mapped reads into 1 Mb genomic windows. We normalized the read counts using two unrelated healthy controls. The resulting ratios were transformed with log2 and adjusted for GC content. The fully normalized log2 ratios were subjected to segmentation using the copy number analysis method. After the segmentation, we selected the most significantly deleted genomic region with segment size &gt;20 Mb in each library pair and calculated ctDNA content using formula: 100 × (1-2log2 ratio). We also calculated the plasma genomic abnormality (PGA) score (a composite score algorithm to reflect overall ctDNA burden in peripheral blood) to estimate ctDNA burden. Overall, we prepared and sequenced a total of 27 libraries including ten dsDNA libraries, ten ssDNA libraries and seven pure-ssDNA libraries, using ten cfDNAs from cancer patients. We received an average of 24264998 raw read sequences and 19495014 mappable reads per sequencing library. Duplicate rate in the ssDNA libraries (mean=0.057%, range=0.046-0.083%) and pure-ssDNA libraries (mean=0.06%, range= 0.054-0.065%) was significantly lower than in dsDNA libraries (mean=0.2%, range=0.14-0.249%) (p&lt;0.001 and p&lt;0.01, respectively), indicating that ssDNA-based library preparation method may better preserve the diversity and complexity of ctDNAs. Library insert size analysis showed that ssDNA libraries were on average 14bp shorter (range from 6 bp to 21 bp) than their matched dsDNA libraries. We also found the Ampure XP beads ratio influenced the library size. We observed consistently higher ctDNA content in ssDNA libraries than in matched dsDNA libraries (p&lt;0.0005). Comparing ssDNA libraries (mean ctDNA content=34%) to their matched dsDNA libraries (mean ctDNA content=30.7%), the increased ctDNA content ranged from 1.3% to 6.4% (mean=3.3%). ctDNA content was also significantly higher in pure-ssDNA libraries than in their matched dsDNA libraries (p&lt;0.005). The increased ctDNA content ranged from 1.0% to 6.9% (mean=3.6%). The PGA scores were always higher in the ssDNA libraries (p&lt;0.0001) or pure-ssDNA libraries (p&lt;0.005) compared to the matched dsDNA method. Our results demonstrate that ssDNA libraries enrich more ctDNA than dsDNA libraries do. Pre-existing ssDNA in plasma is abundant and provides sufficient resource for genomic analysis of ctDNA.

Citation Format: Jing Zhu, Jingyong Huang, Peng Zhang, Manish Kohli, Chiang-Ching Huang, Liang Wang. Advantages of ssDNA over dsDNA library preparation for capturing cell-free tumor DNA in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3536.