WISECONDOR: detection of fetal aberrations from shallow sequencing maternal plasma based on a within-sample comparison scheme

Abstract

Genetic disorders can be detected by prenatal diagnosis using Chorionic Villus Sampling, but the 1:100 chance to result in miscarriage restricts the use to fetuses that are suspected to have an aberration. Detection of trisomy 21 cases noninvasively is now possible owing to the upswing of next-generation sequencing (NGS) because a small percentage of fetal DNA is present in maternal plasma. However, detecting other trisomies and smaller aberrations can only be realized using high-coverage NGS, making it too expensive for routine practice. We present a method, WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR), which detects small aberrations using low-coverage NGS. The increased detection resolution was achieved by comparing read counts within the tested sample of each genomic region with regions on other chromosomes that behave similarly in control samples. This within-sample comparison avoids the need to re-sequence control samples. WISECONDOR correctly identified all T13, T18 and T21 cases while coverages were as low as 0.15-1.66. No false positives were identified. Moreover, WISECONDOR also identified smaller aberrations, down to 20 Mb, such as del(13)(q12.3q14.3), +i(12)(p10) and i(18)(q10). This shows that prevalent fetal copy number aberrations can be detected accurately and affordably by shallow sequencing maternal plasma. WISECONDOR is available at bioinformatics.tudelft.nl/wisecondor.

Figure 1.

Overview of WISECONDOR, showing the data flow starting at the top through all main steps into the classification at the bottom. Note that ‘test samples’ and ‘reference samples’, used to find the reference bins, follow different paths in the data flow. The dashed steps are not part of WISECONDOR and can be interchanged with other mapping strategies, such as BWA (19), BowTie (20), etc.

Figure 2.

Finding within-sample reference bins by WISECONDOR. (a) Shows an area on chromosomes 1 and 2 for two normal (diploid) samples X and Y. The red bar is the target bin (TB) for which a set of reference bins is to be determined. Reference bins are not allowed to be present on the same chromosome. Hence, the set of reference bins for the indicated target bin in this example all need to be on chromosome 2. (b) Squared differences between target bin TB and each of the bins on chromosome 2 for both samples. (c) Summation of the squared differences between target bin TB and each of the bins on chromosome 2 over both samples. Numbers show the similarity ranking of the bins with respect to target bin TB. Red arrows indicate the bins chosen for target bin TB to be included in the set of reference bins. The bin ranked third is not included because it is directly connected to a bin previously selected (bin 2). (d) Stars on each row illustrate selected reference bins on chromosome 2 for every bin of chromosome 1. Notice that the set of reference bins differs for different target bins (rows in this panel).

Figure 3.

Overview of the selected reference bins for all target bins on chromosome 21. Cytobands are shown on the outside of the circle along with base pair positions in Mb and chromosome number. Lines connect target bins on chromosome 21 and their corresponding reference bins on other chromosomes. Opacity indicates the amount of overlapping lines. Barely any connections to chromosome 19 are made as the read frequency behavior of bins on chromosome 19 differs too much from the bins on chromosome 21 over different samples. Repeat rich regions such as the acrocentric p-arm of chromosome 21 and the regions surrounding the centromeres could not be mapped, and will therefore not be selected as reference bins. Image made using Circos (21).

Figure 4.

Samples with trisomies 13, 18, 21 and 22 demonstrate the difference in calling results from the sliding window method and the individual bin method. The vertical axis depicts the z-score, and the horizontal axis the bins on chromosomal positions. The blue line shows the z-score per bin, the red line plots the z-score using the sliding window (). Purple regions show bins called by WISECONDOR (i.e. the sliding window approach). Dark green regions mark bins called with the individual bin method. Light green and pink regions are bins found deviating, by the individual bin and sliding window approach respectively, but those are too small in width such as spikes (and thus did not pass WISECONDOR’s minimum size requirement). Horizontal gray lines denote the abs(z) = 3 threshold. Gray regions are uncallable regions, where light gray is caused by being unable to find enough reference bins and dark gray regions are bins containing mostly unmappable (repetitive) sequences. (a) Sample A5: Trisomy 13. (b) Sample A9: Trisomy 18. (c) Sample A11: Trisomy 21. (d) Sample C18: Trisomy 22.

Figure 5.

Output results for subchromosomal aberration detection (for an explanation of the plots, see Figure 4). (a) Sample C13: isochromosome 12p10. (b) Sample C5: deletion in the middle of the long arm of chromosome 13. (c) Sample C6: loss of the short arm on chromosome 18 (18p10) to the left and the gain of the long arm (18q10) to the right of the centromere. Note the difference in signal height between the two extra copies on chromosome 12p, and one extra copy on chromsome 18q.

Figure 6.

An example of a false negative (for an explanation of the plot see Figure 4). Sample C19: the 7p + [8]/[12] mosaic is not detected (also no considerable deviating z-scores of the bins, as indicated by the blue line, are noticable).

Figure 7.

Examples of false positives (for an explanation of the plot see Figure 4). (a) Sample D11: false positive on chromosome 6. A 2 Mb sized peak is spread out due to the sliding window method. Additional testing proved this was caused by a maternal CNV. (b) Sample C21: false positive on chromosome 19. Note the lack of data points (testable bins) as can be seen from the shape of the blue line and the large amount of gray areas, which is most likely caused by the different GC-content in chromosome 19 compared with other chromosomes.