Single-Stranded DNA Library Preparation Does Not Preferentially Enrich Circulating Tumor DNA

To the Editor: Recent studies have demonstrated that the fragment size of circulating tumor DNA (ctDNA) is shorter than normal cell-free DNA (cfDNA)1 (1, 2). As the most common ctDNA fragment lengths were between 134 bp and 144 bp, whereas the modal cfDNA fragment length was 167 bp (2), it was suggested that the enrichment of shorter plasma fragments might improve analysis of ctDNA. Recently, improved recovery of short plasma DNA fragments by single-stranded DNA (ssDNA) library preparation was reported (3). Therefore, we tested whether ssDNA library preparation results in an enrichment of ctDNA compared to the typically employed double-stranded DNA (dsDNA) libraries. To this end, we applied an adaptor-ligation method as published by Burnham et al. (3) and prepared ssDNA and dsDNA libraries from 6 plasma samples derived from patients with breast (B7\_2, B13\_1), colon (C177\_2, C177\_3), and prostate (P226\_4, P226\_6), all with late-stage metastatic disease. We first tested the fragment length distribution by paired-end massively parallel sequencing. The ssDNA library size distribution profiles exhibited a plateau in their ascending slope caused by short DNA fragments (<150 bp), which was not present in the dsDNA libraries (Fig. 1). These short fragments represented a mean 45.7% of total DNA in all ssDNA libraries (range, 41.9%–56.4%). Hence, the ssDNA libraries confirmed the earlier reported …