Exploring ultrashort populations of circulating nucleic acids by single-stranded plasma DNA sequencing - 11th International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS)

Irena Hudecova ^1,2 Christopher G. Smith ^1,2 James A. Morris ^1,2 Chandra S. Chilamakuri ¹ Katrin Heider ^1,2 Dineika Chandrananda ^1,2 Aadhitthya Vijayaraghavan ^1,2 Simon Pacey ^2,3 Richard Baird ^2,3 Charles E. Massie ³ Elizabeth K. Moore ^1,2,4 James D. Brenton ^1,2,3 Nitzan Rosenfeld ^1,2 Florent Mouliere ^1,2,5

¹Cancer Research UK, Cambridge Institute, Cambridge, UK
²Cancer Research UK, Cambridge Centre, Cambridge, UK
³Department of Oncology, University of Cambridge, Hutchison-MRC Research Centre, Cambridge, UK
⁴Department of Obstetrics and Gynaecology, Addenbrooke’s Hospital, Cambridge, UK
⁵Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

INTRODUCTION

Traditional DNA library preparation protocols recover intact double-stranded DNA (dsDNA), but methods that also capture damaged or single-stranded DNA (ssDNA) offer the opportunity to access a wider spectrum of cell-free DNA (cfDNA) molecules in plasma or other body fluids. Populations of cfDNA recovered through ssDNA-based protocols may provide additional information which can be exploited to study different physiological or pathological conditions.

METHODS

dsDNA- and ssDNA-based protocols, in combination with different DNA extraction methods (column- and magnetic bead-based), were applied to plasma DNA from healthy individuals and patients with multiple cancer types. To assess the differences between these protocols, we explored DNA fragment sizes, and the overall representation of somatic copy number aberrations (SCNA) and single nucleotide variants (SNVs) by shallow whole genome- and whole exome-sequencing respectively. Additionally, we studied the genomic localisation of ultrashort DNA fragments, and explored their relationship with regulatory elements.

RESULTS

ssDNA- and dsDNA-based protocols showed a similar representation of SCNA and SNVs among patients. However, magnetic bead-based DNA extraction coupled with ssDNA library preparation revealed a population of ultrashort DNA fragments centred at 50bp. These showed greater prominence in healthy individuals (median=19%) as compared to cancer patients (median=12%), though we also observed a relationship between the extent of enrichment for ultrashort fragments and SCNA status in cancer patients. Importantly, exploration of ultrashort DNA fragments in healthy individuals and cancer patients revealed differences in plasma DNA coverage at genomic locations associated with regulatory elements.

CONCLUSION

Analysis of ssDNA in plasma of cancer patients showed no apparent enrichment of circulating tumour DNA, confirming previous observations. However, our data demonstrate that ssDNA-based approaches can enhance our current knowledge of cfDNA fragmentation patterns, and by extension, provide a tool to explore underlying mechanisms at play in various physiological and pathological states.