Amplification-free sequencing of cell-free DNA for prenatal non-invasive diagnosis of chromosomal aberrations

• We examined bias in sequencing based karyotyping of maternal cell-free DNA.
• We evaluated both unique molecular identifiers (UMIs) and an amplification-free library preparation method.
• We found that both methods reduce bias.
• These methods enable analysis of all chromosomes.
• These methods can be applied to non-invasive prenatal diagnostics.

Cell-free DNA has been used for fetal rhesus factor and sex determination, fetal aneuploidy screening, cancer diagnostics and monitoring, and other applications. However current methods of using cell free DNA require amplification, which leads to allelic dropout and bias especially when starting with small amounts of DNA. Here we describe an amplification-free method for sequencing of cell-free DNA, even from low levels of starting material. We evaluated this method in the context of prenatal diagnosis of fetal aneuploidy and compared it with a PCR-based library preparation method as well as a recently described method using unique molecular identifiers (UMI). All methods performed well, however coverage was increased by the amplification-free method and GC-induced bias was reduced by both the amplification-free method and the UMI method. Future diagnostic applications including whole genome sequencing of cell-free DNA will benefit from amplification-free sequencing.