Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 58; no. 4; pp. 699 - 706
• Main Authors: VAN DEN OEVER, Jessica M. E, BALKASSMI, Sahila, BOON, Elles M. J, JOANNE VERWEIJ, E, ITERSON, Maarten Van, ADAMA VAN SCHELTEMA, Phebe N, OEPKES, Dick, VAN LITH, Jan M. M, HOFFER, Mariëtte J. V, DEN DUNNEN, Johan T, BAKKER, Egbert
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for Clinical Chemistry 01.04.2012; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Aneuploidy; Biological and medical sciences; Chromosomes; Data processing; Deoxyribonucleic acid; DNA; DNA - blood; DNA - genetics; DNA methylation; DNA sequencing; Down Syndrome - diagnosis; Epigenetics; Female; Fetus; Fetuses; Fundamental and applied biological sciences. Psychology; Genetic testing; Genomes; Gestation; Guanylate cyclase; Humans; Investigative techniques, diagnostic techniques (general aspects); Male; Medical sciences; Methods; Molecular biophysics; Plasma; Polymerase Chain Reaction; Pregnancy; Pregnancy Trimester, First; Retrospective Studies; Risk assessment; Sample preparation; Sequence Analysis, DNA - methods; Trisomy; Womens health; Chromosomal aberration; Trisomy; Nucleotide sequence; Free form; Chromosome G21; Aneuploidy; Biochemistry; Down syndrome; Blood plasma; Molecules; Mother; DNA; Clinical biology; Non invasive method; Diagnosis; Molecular biology; Detection; Sequencing
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2011.174698

Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.