Beyond Aneuploidy Screening: Opportunistic Pathogen Detection from Unmapped Reads in Routine Non-Invasive Prenatal Testing

Background: Routine non-invasive prenatal testing (NIPT) generates tens of millions of sequencing reads per sample, the majority of which are aligned to the human genome, while unmapped reads are typically discarded during the analysis. We investigated whether these otherwise unused reads could be repurposed for maternal pathogen screening. Methods: An in-house bioinformatic pipeline was developed to analyze unmapped reads from NIPT sequencing data for pathogen detection. The pipeline was initially applied to a pilot cohort of 2,000 international prenatal cfDNA datasets, followed by an additional 6,595 samples for expanded analysis. Samples identified as pathogen-positive by NextSeq 500 sequencing were subjected to deep resequencing on the NovaSeq 6000 platform. Analytical sensitivity and the minimum detectable fragment threshold were evaluated using commercially available reference materials for CMV and T. gondii, prepared by serial dilution (0–500 copies) and spiked into sheared human genomic DNA mimicking cfDNA prior to library preparation and sequencing. Results: Across approximately 8,600 clinical sequencing datasets, five pathogens associated with maternal and fetal health risks were detected, with a total of 23 pathogen-positive samples identified based on high-confidence read pairs: cytomegalovirus (CMV, n=13), parvovirus B19 (B19V, n=3), varicella-zoster virus (VZV, n=3), herpes simplex virus (HSV, n=2), and Toxoplasma gondii (T. gondii, n=2). Deep resequencing of six representative pathogen-detected samples yielded a 33- to 239-fold increase in pathogen-derived read pairs compared with the corresponding NextSeq data, providing evidence for the reproducibility of the pathogen-associated signals. Mapping-position analysis showed that the recovered pathogen-associated read pairs were evenly distributed across multiple regions of the corresponding pathogen reference genomes rather than being concentrated within a singlesome specific genomic locius. To establish the analytical detection of the pipeline, limit of detection (LOD) analyses were performed using certified reference materials. Importantly, a practical detection criterion of ≥ 2 high-confidence pathogen-associated read pairs per sample was supported by the complete absence of pathogen-derived reads in all negative controls. The LOD was 250 copies for CMV (100% detection, 20/20 replicates) and as low as 1 copy for T. gondii (95% detection, 19/20 replicates). Conclusions: Human-genome-unmapped reads generated during NIPT sequencing, which are typically excluded from downstream NIPT analysis, contain clinically relevant pathogen-derived DNA fragments. By repurposing these reads, this integrated NGS approach simultaneously screens for fetal chromosomal abnormalities (including aneuploidies and microdeletions/microduplications) and multiple congenital pathogens from a single blood draw, without additional laboratory procedures. These findings expand the clinical utility of prenatal cfDNA sequencing beyond genetic fetal aberrations to the detection of microbial infections, highlighting advances in NIPT testing and its potential for clinical translation.