Abstract

Fetal genetic testing plays a crucial role in  providing insight into fetal health  and informing prenatal or postnatal care. Non-Invasive Fetal Sequencing (NIFS) is an exome sequencing based approach that detects fetal mutations using cell-free DNA (cfDNA) from maternal blood, which avoids the risk of miscarriage associated with invasive procedures. However, the small fraction of fetal cfDNA in maternal blood, known as the fetal fraction (FF), limits the sensitivity of NIFS. This study aims to develop and evaluate a workflow to enrich FF in NIFS using gel electrophoresis-based size selection, leveraging shorter fragment length of fetal cfDNA compared to maternal cfDNA. 

Bovine serum samples were used in preliminary experiments to optimize gel conditions and fragment size windows for selection. Size selection was performed at two stages of the NIFS workflow to determine the optimal conditions: before or after exome capture using automated gel-based system Pippin Prep. Quality control measures using Qubit and TapeStation were performed to assess changes in fragment length, DNA concentration, and total library yield following size selections. Fetal fraction enrichment was subsequently evaluated by sequencing on Illumina NovaSeq X platform and comparing FF estimates before and after size selection. 

Pre-capture size selection produced greater FF enrichment than post-capture selection, while maintaining sufficient yield for downstream capture. Across samples with initial FF of 6.8-41.7%, size selection increased FF by an average of 21% with the greatest improvement observed in samples with a low initial FF (6.8-15%), showing an average increase of 28%. These results suggest that size selection can improve NIFS sensitivity, potentially lowering costs while improving non-invasive prenatal diagnostic accuracy.