Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.
Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.
Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
Philip
P
Burnham
Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
Min Seong
MS
Kim
National Institutes of Health, Bethesda, MD 20892, USA.
Sean
S
Agbor-Enoh
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford CA 94305, USA.
Helen
H
Luikart
National Institutes of Health, Bethesda, MD 20892, USA.
Hannah A
HA
Valantine
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford CA 94305, USA.
Kiran K
KK
Khush
Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
Iwijn
I
De Vlaminck
D000073888:Cell-Free Nucleic Acids / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D015723:Gene Library; D005838:Genotype; D006801:Humans; D016040:Lung Transplantation; D008928:Mitochondria / Q000235:genetics*; D009707:Nucleosomes / Q000235:genetics*; D057089:Pathology, Molecular; D017422:Sequence Analysis, DNA
D000073888:Cell-Free Nucleic Acids; D004277:DNA, Single-Stranded; D009707:Nucleosomes