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.Cell-free DNA (cfDNA) is quickly finding application as a monitoring tool in pregnancy, cancer and organ transplantation [1][2][3][4][5] . cfDNA exists in circulation in many shapes and forms, including fragments of the nuclear genome, the mitochondrial genome and microbial genomes 6 . The predominant type of cfDNA is derived from the nuclear genome and has a fragment size centered around 166 bp, approximately the length of a segment of DNA wound around a histone octamer 7,8 . These nucleosomal fragments of cfDNA are readily accessible for sequencing using standard library preparation methods that are based on ligation of dsDNA sequencing adapters. The most commonly used implementations of this method rely on multiple bead-based size-selective steps that eliminate unwanted adapter-dimer products. These methods, although relevant to a wide range of applications, are not sensitive to the full diversity of circulating DNA 9 ; in particular shorter fragments, highly degraded fragments and (partially) single-stranded fragments of DNA in circulation remain undetected (Fig. 1).An interesting parallel exists with genomic analyses of ancient DNA samples, where the target DNA is usually highly fragmented and present in low amounts 10 . Recently, Meyer et al. introduced a sequencing library preparation method that is based on single-stranded ligation and demonstrated the method by sequencing of the genome of an extinct archaic human 11,12 . Here, we have applied this protocol to sequencing of cfDNA in plasma, motivated by the hypothesis that a library preparation based on ...