Noninvasive fetal genotyping of paternally inherited alleles using targeted massively parallel sequencing in parentage testing cases

Abstract

In our study, we evaluate a straightforward method that can be used to identify paternal alleles based on analyses of paternal alleles and sequencing errors in maternal plasma. Our results support the notion that an MPS-based method could be utilized in noninvasive fetal genotyping and prenatal paternity analyses.

Noninvasive prenatal paternity testing by means of SNP‐based targeted sequencing

“…Following sequencing data analysis, 108 to 174 target‐SNPs were classified as effective‐SNPs and included in paternity calculations (Table ). These numbers were comparable with that reported in a previous study where an initial panel of over 1400 SNPs had been sequenced, but only 130 to 162 SNPs were used in paternity calculations, suggesting that the present selection process effectively reduced the number of redundant SNPs and increased the percentage of effective‐SNPs. Although, in general, the more SNPs sequenced, the more the discriminating power of the test, in practice, the actual number of SNPs sequenced would be limited by costs and high cfDNA input, and the discriminating power would be dependent on the number of SNPs that are eventually included in paternity calculations.…”

“…Importantly, the SNPs selection process significantly reduced the number of sequenced target‐SNPs yet supported comparable discriminating power, and the amplicon‐based approach allowed the use of UMI barcoding to facilitate absolute quantification of SNP alleles and more reliable genotyping, both features representing significant improvements to previous methods. Moreover, where only maternally homozygous SNPs were included in paternity calculations in previous studies, the present analysis algorithm included all SNPs with high‐confidence fetal genotype calls, disregarding the maternal genotype, thus providing an added means to increase the effective‐SNPs percentage. Furthermore, the sequencing of significantly reduced numbers of target‐SNPs associates with reduced costs and increased cost‐effectiveness.…”

“…A major challenge of SNP‐based tests is the accurate genotyping of fetal SNPs in the low fetal fraction (FF; average approximately 10% at 10‐13 gestation weeks) in cfDNA extracted from maternal blood (maternal cfDNA). High‐density array chips and high‐throughput next‐generation sequencing are efficient SNP genotyping platforms, and both have shown success in this application. The use of targeted sequencing (hybridization‐based target enrichment of 5000‐8000 SNPs) and a Bayesian analysis approach successfully determined paternity in 17 clinical cases .…”

Noninvasive prenatal paternity testing using targeted massively parallel sequencing

“…Recent studies based on the Illumina MiSeq platform (Illumina, Inc.) and Ion Torrent PGM platform (Thermo Fisher Scientific) have demonstrated the great potential of the MPS technology in the application of NIPAT. [12][13][14] Meanwhile, several strategies for data interpretation in this field have been also developed. Yang and colleagues 12,14 provided a straightforward nonmaternal allele counting method in maternal plasma for identifying paternal alleles based on a predefined allele fraction cutoff.…”

“…Currently, Illumina sequencers, based on the sequencing by synthesis (SBS) technology, and Ion Torrent sequencers, based on semiconductor technology, have become the predominant MPS platforms widely used in various life science fields. Recent studies based on the Illumina MiSeq platform (Illumina, Inc.) and Ion Torrent PGM platform (Thermo Fisher Scientific) have demonstrated the great potential of the MPS technology in the application of NIPAT . Meanwhile, several strategies for data interpretation in this field have been also developed.…”