Creating basis for introducing non‐invasive prenatal testing in the Estonian public health setting

Abstract

Objective The study aimed to validate a whole‐genome sequencing‐based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. Method In total, 424 maternal blood samples were included. Analysis pipeline involved cell‐free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre‐defined per‐chromosome sets of unique k‐mers from sequencing raw data. SeqFF was implemented to estimate cell‐free fetal DNA (cffDNA) fraction. Results NIPTmer identified correctly all samples of non‐mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false‐positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid. Conclusion We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination.

Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

“…As library preparation time for both MPS- and TOP-seq-based protocols is comparable (up to 6 h), the number of required sequencing reads highlights the possible cost-effectiveness of the TOP-seq approaches. In contrast to whole-genome shallow coverage NIPT that requires approximately 10–20 million reads [ 37 , 38 ] for high-quality karyotyping, TOP-seq allows accurate prediction of fetal karyotype with as low as 1 million single-end reads. Another advantage of TOP-seq is the simplicity of data analysis.…”

“…Seven of these women were carrying fetuses with trisomy 21. All samples were obtained from the pregnant women with the gestational age of 12-20 weeks with singleton pregnancy and undergoing the low-coverage NIPT whole-genome MPS sequencing using Illumina technology as previously described [37,42]. SeqFF was used for calculating the cell-free fetal DNA fraction for all the samples [27].…”

Systematic evaluation of NIPT aneuploidy detection software tools with clinically validated NIPT samples

“…The validation sample set was based on a previously published validation study by Žilina et al . (14), consisting of 423 samples, of which 258 were high-risk pregnancies that had undergone diagnostic invasive prenatal analysis (14). These included 19 samples with confirmed fetal T21, eight T18 and three T13 samples.…”

“…All samples were sequenced with Illumina NextSeq 500 platform, producing 85 bp single-end reads with an average per-sample coverage of 0.32× at the University of Tartu, Institute of Genomics Core Facility, according to the manufacturer’s standard protocols, as described previously (14). This study was performed with the informed consent from the participants and with approval of the Research Ethics Committee of the University of Tartu (#315/T-13).…”

“…Pre-built lists, which were packaged with the software, were used. Although the cleaned lists were made with the GRCh37 reference genome, since it is not an alignment-based method and GRCh37 lists are known to work (14), the GRCh37 version was kept. Binaries were updated to the latest version due to software issues with the packaged binary.…”