Comparison of Single Molecule, Real-Time Sequencing and Nanopore Sequencing for Analysis of the Size, End-Motif, and Tissue-of-Origin of Long Cell-Free DNA in Plasma

Background Recent studies using single molecule, real-time (SMRT) sequencing revealed a substantial population of analyzable long cell-free DNA (cfDNA) in plasma. Potential clinical utilities of such long cfDNA in pregnancy and cancer have been demonstrated. However, the performance of different long-read sequencing platforms for the analysis of long cfDNA remains unknown. Methods Size biases of SMRT sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies (ONT) were evaluated using artificial mixtures of sonicated human and mouse DNA of different sizes. cfDNA from plasma samples of pregnant women at different trimesters, hepatitis B carriers, and patients with hepatocellular carcinoma were sequenced with the 2 platforms. Results Both platforms showed biases to sequence longer (1500 bp vs 200 bp) DNA fragments, with PacBio showing a stronger bias (5-fold overrepresentation of long fragments vs 2-fold in ONT). Percentages of cfDNA fragments > 500 bp were around 6-fold higher in PacBio compared with ONT. End motif profiles of cfDNA from PacBio and ONT were similar, yet exhibited platform-dependent patterns. Tissue-of-origin analysis based on single-molecule methylation patterns showed comparable performance on both platforms. Conclusions SMRT sequencing generated data with higher percentages of long cfDNA compared with nanopore sequencing. Yet, a higher number of long cfDNA fragments eligible for the tissue-of-origin analysis could be obtained from nanopore sequencing due to its much higher throughput. When analyzing the size and end motif of cfDNA, one should be aware of the analytical characteristics and possible biases of the sequencing platforms being used.

Automated summary

Recent studies have highlighted the presence of a substantial amount of analyzable long cell-free DNA (cfDNA) in plasma through single molecule, real-time (SMRT) sequencing. In this study, the size biases of SMRT sequencing by PacBio and nanopore sequencing by ONT were evaluated. The results showed that both platforms had biases towards sequencing longer DNA fragments, with PacBio showing a stronger bias. Tissue-of-origin analysis based on single-molecule methylation patterns performed comparably on both platforms. It is important to be aware of the analytical characteristics and biases of the sequencing platforms used when analyzing the size and end motif of cfDNA.