High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

Abstract

BackgroundCirculating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.MethodsWhole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.ResultsTwo parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.ConclusionsThese data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women

“…The cffDNA in maternal peripheral blood consists of small fragments that are unstable, and its concentration reduces at a constant speed after collecting blood because of the activity of DNases. It is difficult to extract cffDNA from maternal peripheral blood after 6 hours (Jensen et al, 2013;Wong et al, 2013). Thus, we isolated the cffDNA immediately after collecting the blood samples to avoid DNA degradation.…”

Prenatal diagnosis of congenital adrenal hyperplasia owing to 21-hydroxylase deficiency

“…This focused treatment cannot be accomplished through genetic diagnosis of CAH by amniocentesis and chorionic villus sampling. It has been shown that detection of fetal chromosomal aneuploidy from cffDNA in the maternal plasma using MPS has a high sensitivity and specificity leading to its commercial use [31][32][33]. While our data are promising, the number of studied cases is still relatively small, requiring further validation in large-scale prospective studies.…”

Noninvasive Detection of a Balanced Fetal Translocation from Maternal Plasma

“…The most established methods have used MPS to detect certain autosomal trisomies, sex chromosome aneuploidies, and other copy number variations (CNVs) with high analytical sensitivity and specificity (1)(2)(3)(4)(5)(6). In addition, the utility of sequencing ccf DNA has been further demonstrated by detecting mutations associated with Mendelian traits and the reconstruction of the entire fetal genome (7)(8)(9)(10).…”