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

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Abstract

Background

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

Objective

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

Methods

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.

Results

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.

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.

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Acknowledgments

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).

Author information

Authors and Affiliations

  1. Laboratory of Medical Genetics, Harbin Medical University, and The Key Laboratory of Preservation of Human Genetic Resources and Disease Control in China, Chinese Ministry of Education, 157 Baojian Road, Harbin, 150081, Heilongjiang, China

    Jing Zhu

  2. Department of Pathology and MCW Cancer Center, Medical College of Wisconsin, Milwaukee, WI, USA

    Jinyong Huang, Peng Zhang, Qianxia Li & Liang Wang

  3. Department of Genitourinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

    Manish Kohli

  4. Joseph J. Zilber School of Public Health, University of Wisconsin, Milwaukee, WI, USA

    Chiang-Ching Huang

  5. Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Dr, Tampa, FL, 33612, USA

    Liang Wang

Authors
  1. Jing Zhu
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  2. Jinyong Huang
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  3. Peng Zhang
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  4. Qianxia Li
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  5. Manish Kohli
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  6. Chiang-Ching Huang
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  7. Liang Wang
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Corresponding authors

Correspondence to Jing Zhu or Liang Wang.

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Funding

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.

Electronic supplementary material

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Supplementary Figure S1

Correlation between insert size difference and ctDNA burden difference. For ssDNA and their matched dsDNA libraries, a positive correlation (r = 0.673, p = 0.017) was found between insert size difference and ctDNA burden difference

Supplementary material 2 (XLSX 9 kb)

Supplementary material 3 (XLSX 9 kb)

Supplementary material 4 (XLSX 9 kb)

Supplementary material 5 (XLSX 9 kb)

Supplementary material 6 (XLSX 18 kb)

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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). https://doi.org/10.1007/s40291-019-00429-7

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  • DOI: https://doi.org/10.1007/s40291-019-00429-7

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