A review on the impact of single-stranded library preparation on plasma cell-free diversity for cancer detection

Abstract

In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNA-LP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors.

Keywords: cell-free DNA; fragment-size; liquid biopsy; single-stranded library preparation; ultrashort single-stranded cell-free DNA.

Figure 1

Double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) library preparation incorporates different DNA species from cell-free DNA. (A) Principal differences between the two library preparation methodologies. The initial heat-denature step in single-stranded library preparation allows the inclusion of multiple conformations of cell-free DNA. (B) Representative fragment profiles generated by double-stranded and single-stranded library preparations show that single-stranded library preparations are more sensitive for representing shorter cfDNA fragments below 80bp. Data has been derived from (11, 21).

Figure 2

Different strategies to append the initial adapter to a single-stranded molecule in a single-stranded DNA library preparation workflow. Schematic diagrams for the (A) Terminal Deoxynucleotidyl Transferase (TDT) - Mediated Ligation, (B) RNA Ligase, (C) Splint Adapter, and (D) TDT-assisted adenylate connector-mediated single-stranded (ss) DNA (TACS) methods are shown.

Figure 3

Schematic diagram showing that pairing various low molecular weight enriched extraction methods with ssDNA library preparation reveals the presence of ultrashort single-stranded cell-free DNA in plasma. Data has been derived from (11, 21).

Figure 4

Unique properties of plasma ultrashort single-stranded cell-free DNA (uscfDNA). (A) Digestion assays suggest ultrashort cell-free DNA is single-stranded. (B) Peak detection bioinformatic tools indicate that uscfDNA maps as abundant peaks along the genome, and these peaks are enriched in (C) regulatory regions. (D) Sequences of uscfDNA contain potential G-Quad secondary structures. Data has been derived from (11, 21).