A50: Sensitive Mutation Detection By Sequencing Circulating Cell-Free DNA

Nan Fang1,Rumeysa Tolun1,Katja Heitz1,Colin Baron1

1QIAGEN GmbH, Hilden, Germany

Presenting date: Monday 2 November
Presenting time: 13.10-14.00


Circulating DNA, the cell-free DNA (cfDNA) in serum or plasma, has become a powerful tool used for Non-Invasive Prenatal Testing (NIPT) as well as cancer liquid biopsy.  It has been shown that the quantity, integrity, as well as the mutation contents of the cfDNA in cancer patients could differ from that in healthy controls and therefore serve as biomarkers for cancer diagnosis, prognosis, and stratification.  High-throughput sequence analysis of the cfDNA with the rapidly developing next generation sequencing (NGS) technologies provides a highly sensitive method in detecting and characterizing somatic mutations in the cancer patients. However, the concentration of the cfDNA in the serum is normally very low, which posts challenges in sequencing library construction.


We tested different parameters that can affect the efficiency of cell free DNA library construction, such as adaptor concentration, size selection parameters, and selection of the enzymes and master mixes.


We developed an optimized workflow that combines high-efficiency NGS library construction, unbiased library amplification, and target enrichment to sensitively and reliably detect mutations in cfDNA samples.


Efficient adaptor ligation, adaptor dimer removal, and library amplification all contribute to the sensitivity of the mutation detection in cell free DNA sequencing. A reliable mutation detection algorithm is also required for sensitive NGS-based mutation calling.