Development of a new methylation‐based fetal fraction estimation assay using multiplex ddPCR

Abstract

Background: Non-invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS-based methods can reliably detect fetal aneuploidies non-invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal-specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma. Methods: We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non-pregnant female tissues to identify fetal-specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples.Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent. Results: In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM-based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80-0.91). The model was confirmed using an independent set of 53 male pregnancies. Conclusion: By employing a set of well-characterized DMRs, we developed a SNP-, sex-and ploidy-independent methylation-based multiplex ddPCR assay for accurate fetal fraction estimation. K E Y W O R D Sdifferentially methylated regions, fetal fraction estimation, multiplex ddPCR, non-invasive prenatal testing 2 of 10 |

Critical appraisal of droplet digital polymerase chain reaction application for noninvasive prenatal testing

“…Currently various types of digital PCR approaches are available commercially viz. technique based on microfluidic chamber to split the sample into hundreds of nanoliter partition, 27 BEAMing approach, based on either emulsion PCR or target template amplified in the bead and methylation specific multiplex dDPCR approach 14 . ( Table 1 ).…”

“…Several different approaches using massively parallel sequencing (MPS) also called NGS, digital polymerase chain reaction (dPCR) or BEAMing digital PCR or droplet digital PCR (ddPCR) and methylation-specific PCR have been utilized for assessing the fetal fraction as part of the NIPS/NIPD workflow. [14][15][16][17][18][19] Among these, whole-genome sequencing or targeted-sequencing methods by NGS are considered accurate and sensitive, but are both costly and necessitate complex bioinformatics algorithms. The ddPCR is a novel technique that is frugal, equally sensitive, less labor intensive, less time-consuming and plain algorithm dependent method for detecting cffDNA fraction.…”

Fragmentomic cfDNA Patterns in Noninvasive Prenatal Testing and Beyond

“…A combination of these strategies has recently allowed for a methylation-based estimation of the fetal fraction. 69 Methylation-sensitive digestion of the cfDNA pool, in which methylated nucleotides are protected from degradation, depletes abundant hypo-methylated maternal sequences, allowing to increase the fraction of cffDNA. Hyper-methylated fetoplacental genomic loci, such as RASSF1A, SOX14 and TBX3, may serve as useful marker candidates in such enrichment approaches.…”

Self-Internal-Reference Probe System for Control-Free Quantification of Mutation Abundance

“…High-throughput sequencing and droplet digital PCR are absolute quantitative methods. High-throughput sequencing directly calculates the mutation abundance by reading the mutant and normal bases. − Droplet Digital PCR can evaluate the absolute quantity of mutant DNA and WT DNA at the single-molecule level, ,, and thus the mutation abundance can also be directly calculated. Absolute quantification is advantageous; it can confirm whether mutations are present.…”