An SNP panel for the analysis of paternally inherited alleles in maternal plasma using ion Torrent PGM

Abstract

Researchers have sought to develop an effective protocol for paternity analysis using cell-free DNA (cfDNA) in maternal plasma. The use of massively parallel sequencing (MPS) technology for SNP testing is attractive because of its high-throughput capacity and resolution to single-base precision. In this study, we designed a customized SNP panel for cfDNA sequencing that includes 720 short amplicons (< 140 bp) targeting SNPs on the autosome and Y chromosome. The systemic performance was evaluated using the Ion Torrent PGM, indicating balanced coverage among most of the included loci, except for 78 poorly performing SNPs that were observed to have an inconsistent allele balance, lower coverage reads or high background signals. Then, the custom panel was used to perform cfDNA genotyping in maternal plasma from 20 pregnancies in the first and second trimesters (9 to 21 weeks). By establishing an allele fraction cutoff of 2.0%, 53 to 128 autosomal SNP loci were considered informative for paternal origin. Validation results in foetal samples showed that 49.43% to 100% of the real paternal alleles were accurately identified, with incorrect alleles encountered in 3 cases. The concentration of foetal cfDNA ranged from 4.28% to 10.70%. Our results show that this amplicon-based sequencing strategy could be utilized in analysing paternally inherited alleles in maternal plasma. However, further studies and optimization are required for a more detailed and accurate interpretation of the cfDNA sequencing results based on MPS technology.

A microhap panel for kinship analysis through massively parallel sequencing technology

“…However, it seemed that more markers still needed to be added for the identification of second‐degree or more distant relatives to provide more discriminating power. It should also be noted that though the LR value of individual MH locus was lower than STRs on average, the larger number in the human genome, less artifacts (e.g., stutter peaks) and the shorter length of candidate fragment (restricted within ∼100 bp in the present study) are expected to make this maker more useful in cases of unbalanced mixed sample or degraded DNA, such as the DNA mixture in a crime scene or the cell‐free fetal DNA utilized in the prenatal paternity determination .…”

“…Altogether, 60 MH candidates were primarily screened out from ALFRED database (Allele Frequency Database, Yale University) and previous published literatures (Supporting Information ). Altogether, there were 46 3‐SNPs, 13 4‐SNPs, and 1 5‐SNPs MHs in the candidate panel, spreading across 21 human autosomes.…”

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.…”

“…[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. Validation of this method in fetal tissues demonstrated that paternal alleles were accurately identified.…”

“…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.…”