Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms

PLoS One. 2017 Oct 30;12(10):e0186771. doi: 10.1371/journal.pone.0186771. eCollection 2017.


Objective: Cell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction.

Methods: A multiplex panel of primers was designed for 35 indels plus a ZFX/ZFY amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA.

Results: Using gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)).

Conclusions: Using our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies.

MeSH terms

  • Cell-Free Nucleic Acids / blood*
  • Chromosomes, Human, Y
  • DNA / blood*
  • Female
  • Fetus / metabolism*
  • Humans
  • INDEL Mutation*
  • Pregnancy


  • Cell-Free Nucleic Acids
  • DNA

Grant support

We acknowledge the National Medical Research Council/Clinician Scientist Award (NMRC/CSA/0059/2014) for supporting our work. The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.