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.
Highlights
Since the discovery that fetal DNA comprises 3–20% of circulating cell-free DNA in maternal plasma [1,2,3], the utilization of ccf DNA as an analyte for diagnostic purposes has been increasingly recognized as a powerful noninvasive alternative for aneuploidy detection during pregnancy
Regionspecific methods theoretically support higher throughput while still achieving acceptable performance for the two most common trisomies; these targeted assays, including those not based on Massively Parallel Sequencing (MPS) [10,11,12], are restricted by the significant amount of redevelopment required when additional content, for example less frequent trisomies or sex chromosome aneuploidies, is introduced
Improvements focused on three aspects: I) optimizing library preparation to enable robust yield and increased throughput; II) increasing the number of individually molecularly indexed samples pooled together in a single flowcell lane; and III) improving analytical methods for aneuploidy classification
Summary
Since the discovery that fetal DNA comprises 3–20% of circulating cell-free (ccf) DNA in maternal plasma [1,2,3], the utilization of ccf DNA as an analyte for diagnostic purposes has been increasingly recognized as a powerful noninvasive alternative for aneuploidy detection during pregnancy. Regionspecific methods theoretically support higher throughput while still achieving acceptable performance for the two most common trisomies (trisomy 21 and trisomy 18); these targeted assays, including those not based on Massively Parallel Sequencing (MPS) [10,11,12], are restricted by the significant amount of redevelopment required when additional content, for example less frequent trisomies or sex chromosome aneuploidies, is introduced. It remains to be seen how well these targeted methods can identify events such as partial trisomies or other large copy number variations. 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
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