High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma

Abstract

Background: Circulating 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.

Methods: Whole 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.

Results: Two 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.

Conclusions: These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Figure 1. Library preparation optimization.

A) The standardized library concentration was compared between semi-automated (n = 287) and manual library preparation methods. B) GCRM based z-scores are shown for each of 93 samples. Confirmed euploid samples (n = 83) are shown in gray; confirmed trisomy 21 samples (n = 10) are shown in blue.

Figure 2. Paired comparison of z-scores.

Z-scores were calculated for paired samples with previously described GC normalized, repeat masked z-scores on the x-axis and z-scores from the same libraries sequenced in 12-plex on the y-axis. Samples classified by karyotype analysis as trisomies for A) Chromosome 21, B) Chromosome 13, or C) Chromosome 18 are shown in blue; unaffected samples for each aneuploidy condition are shown in gray. Red horizontal and vertical lines in each plot represent the respective classification cutoff for that chromosome (z = 3 for chromosome 21, z = 3.95 for chromosomes 13 and 18).

Figure 3. Z-score is linked to fetal fraction.

The chromosome specific z-score for each aneuploid chromosome is plotted against the proportion of fetal DNA (fetal fraction). Samples classified by karyotype analysis as trisomies for A) Chromosome 21, B) Chromosome 13, or C) Chromosome 18 are shown in blue; unaffected samples for each aneuploidy condition are shown in gray. Black horizontal line in each plot represents the respective classification cutoff for each chromosome (z = 3 for chromosome 21, z = 3.95 for chromosomes 13 and 18). Dashed blue line in each panel represents a robust linear fit of aneuploid samples. Dashed gray line in each panel represents a robust linear fit of all unaffected samples.

Figure 4. Paired comparison of z-scores.

Z-scores were calculated for 1269 paired samples with previously described GC normalized, repeat masked z-scores on the x-axis and z-scores from the high-throughput assay on the y-axis. Samples classified by karyotype analysis as trisomies for A) Chromsome 21, B) Chromosome 13, or C) Chromosome 18 are shown in blue; unaffected samples for each aneuploidy condition are shown in gray. Red horizontal and vertical lines in each plot represent the respective classification cutoff for that chromosome (z = 3 for chromosome 21, z = 3.95 for chromosomes 13 and 18). Black line in plot represents y = x.