Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA

Abstract

Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

Keywords: NIPT; cell-free DNA; maternal plasma DNA; noninvasive prenatal testing; semiconductor sequencing.

Fig. 1.

Average absolute Z scores of subchromosomal abnormalities for all mixed abnormal and normal DNA samples at various concentrations and sequencing depths. Error bars represent ±1.

Fig. S1.

Characteristic Z scores of a section of DNA encompassing a deletion in sample 14,945 at (A) varying concentrations of abnormal DNA at 5 million reads and (B) varying sequencing depths at 15% concentration of abnormal DNA. (C) The aCGH plot of sample 14,945 is shown for comparison.

Fig. 2.

Smallest size of deletions/duplications detected in mixed abnormal and normal DNA samples at various sequencing depths and concentrations of abnormal DNA.

Fig. 3.

Size distribution of maternal plasma cfDNA fragments sequenced by SSP in a population of pregnant women. The blue region encompasses 130–140 bp (region A), and the red region encompasses 155–175 bp (region B). (A) Representative examples from maternal plasma with different fetal DNA fractions. (B) Aggregate of all samples. The blue line represents the mean read ratio of all samples, and the gray region represents ±1 SD.

Fig. 4.

Estimation of the fetal DNA concentration in maternal plasma using the size distribution of cfDNA fragments. (A and B) Correlation between the reads ratio of DNA fragments in (A) the 130- to 140-bp region or (B) the 155- to 175-bp region and the fetal DNA concentration predicted by the Z score of Y chromosome. (C) Correlation between fetal DNA concentration estimated by regions A and B.

Fig. S2.

Distribution of fetal DNA concentrations estimated by NIPT of maternal plasma in a cohort of 1,476 pregnant women at various gestational ages.