Size-Selection Is Essential for Cell-Free DNA Studies

Advances in using cell-free DNA (cfDNA) to glean clinically meaningful information for a patient have been stunning. For the most part, these research studies (or downstream diagnostic tests) isolate fetal DNA in the mother’s blood or tumor-derived DNA from the background of healthy DNA in the bloodstream. Typically known as liquid biopsies, these minimally invasive assays have been used to detect cancer in asymptomatic patients and to diagnose genetic defects in utero.

DNA Size Is Important for cfDNA Analysis

The increasing use of cell-free DNA for prenatal and cancer applications has put a spotlight on the need for protocols that isolate only the DNA of interest. Many studies have found that accurate size selection is essential for analyzing cell-free DNA, which tends to be shorter for targets like fetal or tumor DNA. One early study found that fetal DNA fragments were shorter than 300 bp while maternal DNA fragments were longer than 1 kb. Another study noted a peak of about 160 bp for fetal DNA.

The Need for DNA Size-Selection Protocols

Cell-free DNA studies are still so nascent that scientists have not yet coalesced around any single protocol as consistently better than the others.  In one study, scientists evaluated methods for assessing fragment size of cell-free fetal DNA. They determined that paired-end sequencing and basic gel electrophoresis were both adequate for distinguishing fetal from maternal DNA. In cases in which they had quite specific fragment sizes, the scientists detected cases of chromosomal aneuploidy simply by noting size aberrations in the fragments. A separate review also walked through size measurement methods for cell-free DNA studies, including protocols such as qPCR and electrophoresis. Another study considered various approaches to analyzing circulating tumor DNA. They reported that selecting for shorter DNA fragments “substantially increased” the detection of an allele frequency of a mutation indicative of lung cancer.

Challenges with Size Selection

Incorporating a size-selection step to enrich for the DNA of interest is a promising concept, but experimentally it faces challenges on the yield front. In most studies, there is precious little cell-free DNA in a blood or plasma sample. Size-selecting with tools that may have low recovery rates is challenging for scientists who are reluctant to diminish viable target DNA even more. However, progress is being made. For example, a poster presentation from the 2016 AGBT Precision Medicine conference demonstrated that pairing technologies — in this case, electrophoresis-based size selection with low-input library prep — can deliver reliable results, even when working with less than 1 ng of size-selected material. With so much interest in cell-free DNA, we will no doubt hear much more about evolving methods for size selection, quantification, and analysis in the coming months.