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2023 ACMG Annual Clinical Genetics Meeting Digital ...
A novel, iteratively designed and highly multiplex ...
A novel, iteratively designed and highly multiplexed droplet digital PCR assay for non-invasive prenatal screening
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Pdf Summary
Researchers from Bio-Rad Laboratories have developed a novel and highly multiplexed droplet digital PCR (ddPCR) assay for non-invasive prenatal screening (NIPS). NIPS is used to determine the risk of fetal abnormalities by testing circulating cell-free DNA (cfDNA) in the maternal bloodstream. While next-generation sequencing (NGS) is the current method of choice, ddPCR offers a faster and more cost-effective alternative.<br /><br />The researchers used methylation-sensitive restriction enzymes (MSREs) to distinguish between fetal and maternal cfDNA. MSRE digestion was performed in-droplet, without disrupting the ddPCR workflow. The fetal fraction, the proportion of fetal DNA in maternal blood, was calculated using the ddPCR assay with the formula provided. Fetal and maternal cfDNA were quantified simultaneously in the same reaction.<br /><br />The researchers used methylation whole-genome sequencing data to design the assay and target regions that showed a high contrast in methylation patterns between fetal and maternal cfDNA. They developed a highly multiplexed assay for ddPCR by combining multiple strategies, including in-house primer design, a universal probe approach, and extensive wet lab testing.<br /><br />The researchers also implemented the PREX primer design engine for high-level multiplexing, minimizing off-target and heterodimer events. The performance of the multiplex assay was evaluated by separating positive and negative droplets in the ddPCR plots, as well as by evaluating the relative concentration of targets in clinical and contrived samples.<br /><br />The results showed that the multiplex assay performed well in detecting aneuploidies, with good classification performance and high sensitivity and specificity. The fetal fraction measurements obtained from the ddPCR assay correlated with those obtained from NGS analysis.<br /><br />Overall, this novel and highly multiplexed ddPCR assay shows promise for accurate non-invasive prenatal screening and can potentially be used in other medical screening areas that require highly multiplexed targeted assay sets.
Asset Subtitle
Presenting Author - Madhumita Ramesh, MS; Co-Author - Todd Warczak, PhD; Co-Author - Nyaradzo Dzvova, PhD; Co-Author - Raymond Abayan, BS; Co-Author - Maria F. Gencoglu, PhD; Co-Author - Thea Riel, BS; Co-Author - Anthony Henriquez, MS; Co-Author - Preetha Markose, MS; Co-Author - Kristin Loomis, PhD; Co-Author - Olga Mikhaylichenko, PhD; Co-Author - Nathan L. Hendel, PhD; Co-Author - Eric Hall, PhD; Co-Author - Richard Dannebaum, PhD; Co-Author - Séverine Margeridon, PhD; Co-Author - Monica Herrera, PhD;
Meta Tag
Bioinformatics
Cell free DNA/cfDNA
Chromosomal Abnormalities
Genome sequencing
Maternal Serum Screening
NextGen Sequencing
Noninvasive prenatal screening (NIPS)
Co-Author
Todd Warczak, PhD
Co-Author
Nyaradzo Dzvova, PhD
Co-Author
Raymond Abayan, BS
Co-Author
Maria F. Gencoglu, PhD
Co-Author
Thea Riel, BS
Co-Author
Anthony Henriquez, MS
Co-Author
Preetha Markose, MS
Co-Author
Kristin Loomis, PhD
Co-Author
Olga Mikhaylichenko, PhD
Co-Author
Nathan L. Hendel, PhD
Co-Author
Eric Hall, PhD
Co-Author
Richard Dannebaum, PhD
Co-Author
Séverine Margeridon, PhD
Co-Author
Monica Herrera, PhD
Presenting Author
Madhumita Ramesh, MS
Keywords
Bio-Rad Laboratories
droplet digital PCR
non-invasive prenatal screening
circulating cell-free DNA
methylation-sensitive restriction enzymes
fetal fraction
methylation patterns
multiplex assay
PREX primer design engine
aneuploidies
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