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



Single-stranded DNA (ssDNA) libraries have been shown to enrich shorter and more degraded DNA fragments than double-stranded DNA (dsDNA) libraries.


In this study, we evaluated whether ssDNA libraries captured more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA).


We prepared dsDNA, ssDNA and pure-ssDNA (capture the preexisting ssDNA) libraries using ten plasma cfDNA samples. After low-pass whole genome sequencing, we calculated a duplicate rate to estimate library complexity and compared the library insert sizes between the different library methods. Finally, we estimated the ctDNA content and plasma genomic abnormality (PGA) score, an indicator of ctDNA burden.


27 libraries were prepared and sequenced from the ten cfDNA samples. The duplicate rate in the ssDNA and pure-ssDNA libraries was significantly lower than in the dsDNA libraries (p < 0.001 and p < 0.01, respectively). ctDNA content and PGA scores were consistently higher in the ssDNA and pure-ssDNA libraries than in the matched dsDNA libraries (p < 0.005). The higher ctDNA content in ssDNA libraries was associated with smaller library insert size.


ssDNA libraries preserve more diversity and capture more ctDNA than dsDNA libraries. The ssDNA library method is preferred when performing genomic analysis of ctDNA.

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The authors thank Molecular Pathology Core in Medical College of Wisconsin for timely sequencing service. We acknowledge the scholarship from the China Scholarship Council (CSC) (201508230031 to JZ).

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Correspondence to Jing Zhu or Liang Wang.

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This research was supported by the National Institute of Health (R01CA212097) to LW; the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2017056), Natural Science Foundation of Heilongjiang Province (YQ2019H002) and the Fund Program of Heilongjiang Province for Selected Returned Overseas Professionals to JZ.

Conflict of Interest

JZ, JH, PZ, QL, MK, CH and LW have no conflicts of interest.

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Zhu, J., Huang, J., Zhang, P. et al. Advantages of Single-Stranded DNA Over Double-Stranded DNA Library Preparation for Capturing Cell-Free Tumor DNA in Plasma. Mol Diagn Ther 24, 95–101 (2020).

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