Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA
Authors: Zhang Daniel Danxu , Zhang Kang 
Published: November 2023

Non‐invasive prenatal testing (NIPT) using sequencing of fetal cell‐free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and has become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high‐risk fetuses. We first demonstrated that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1476 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 (3.5M) reads, SSP detected 56/78 (71.8%) of subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10M reads and restricting the size of abnormalities to more than 1Mb, sensitivity improved to 69/73 (94.5%). Of the 55 false positive samples, 35 were due to deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study demonstrates 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.

Comparison of NIPT and aCGH in clinical samples

Sample ID
dup/del
aCGH Result
Size (Mp)
Syndrome associated with region*
NIPT result

Location predicted by aCGH
FC%
Location predicted by NIPT
3M reads
5M reads
10M reads
15M reads

A0587
del
chr16:29673954‐30198600
0.52
16p11.2 microduplicatoin syndrome
17.01
‐‐
No
No
No
No

A0133
dup
chr19:327273‐863312
0.54

2.01
‐‐
No
No
No
No

A0553
del
chr19:3806228‐4343253
0.54

11.61
‐‐
No
No
No
No

A0519
del
chr3:27275‐853692
0.83
3p syndrome
9.57
‐‐
No
No
No
No

A0957
del
chr7:157683596‐158602499
0.92
7q36.1‐36.3 Deletion
10.34
‐‐
No
No
No
No

A0281
del
chr17:674618‐1859251
1.18
17p13.1 deletion syndrome
9.76
‐‐
No
No
No
No

A1005
dup
chr22:22069437‐23318455
1.25
22q11.2 deletion syndrome
19.82
chr22:22000000‐23000000
No
No
Yes
Yes

A1102
dup
chr2:88015932‐89387655
1.37

20.53
chr2:88000000‐90000000
Yes
Yes
Yes
Yes

A1444
del
chr17:72364514‐73777326
1.41
17q25 Duplication
16.93
chr17:71000000‐73000000
No
No
Yes
Yes

A0187
del
chrX:6500000‐8000000
1.5
Steroid sulphatase deficiency (STS)
NA
chrX:5000000‐7000000
Yes
Yes
Yes
Yes

A0197
dup
chrX:6500000‐8000000
1.5
Steroid sulphatase deficiency (STS)
9.61
chrX:6000000‐7000000
Yes
Yes
Yes
Yes

A0403
del
chrX:6490000‐8040000
1.55
Steroid sulphatase deficiency (STS)
10.58
chrX:5000000‐7000000
Yes
Yes
Yes
Yes

A1042
dup
chr18:587754‐2180664
1.59
18p11.32‐p11.31 duplication
18.42
chr18:0‐2000000
No
No
Yes
Yes

A1138
dup
chr18:36319629‐37915204
1.60
18q12.2‐21.1 deletion
17.27
chr18:35000000‐37000000
No
No
Yes
Yes

A0495
dup
chr16:14408492‐16089759
1.68
16p13.11 microdeletion
20.54
chr16:14000000‐16000000
Yes
Yes
Yes
Yes

A1285
del
chr17:31461588‐33242217
1.78

21.65
chr17:31000000‐33000000
No
No
Yes
Yes

A0844
dup
chr8:1.44E+08‐1.46E+08
1.85
8q22.1‐qter duplication syndrome
10.42
‐‐
No
No
No
No

A1034
del
chr4:27770182‐29952659
2.18
14q11‐q22 deletion syndrome
15.77
chr4:27000000‐29000000
No
No
Yes
Yes

A0248
del
chr4:91399112‐93621545
2.22

25.90
chr4:90000000‐92000000
Yes
Yes
Yes
Yes

A0129
dup
chr4:1.57E+08‐1.60E+08
2.42
4q32.1‐q32.2 triplication syndrome
14.98
chr4:157000000‐159000000
Yes
Yes
Yes
Yes

A0769
del
chr22:17299942‐19770514
2.47
DiGeorge syndrome/22q11.2 deletion syndrome
NA
‐‐
No
No
No
No

A0286
dup
chr22:18919942‐21440514
2.52
DiGeorge syndrome/22q11.2 deletion syndrome
13.05
chr22:20000000‐22000000
Yes
Yes
Yes
Yes

A0686
del
chr6:170426‐2753293
2.58
6pter‐p24 deletion syndrome
NA
chr6:0‐3000000
No
No
Yes
Yes

A0901
del
chr13:65054495‐67810584
2.76

28.55
chr13:65000000‐67000000
No
No
Yes
Yes

A0786
dup
chr21:44077514‐46847409
2.77

22.78
chr21:44000000‐47000000
No
No
Yes
Yes

A0301
del
chr2:1.72E+08‐1.75E+08
3

31.85
chr2:171000000‐174000000
Yes
Yes
Yes
Yes

A0310
del
chr22:17096855‐20311763
3.21
DiGeorge syndrome/22q11.2 deletion syndrome
14.74
chr22:26000000‐30000000
Yes
Yes
Yes
Yes

A0580
dup
chr11:1.31E+08‐1.35E+08
3.39
Jacobsen syndrome
8.68
chr11:131000000‐135000000
No
No
Yes
Yes

A1052
dup
chrY:6688691‐10511314
3.82

17.85
chrY:6000000‐10000000
Yes
Yes
Yes
Yes

A0001
del
chr1:814245‐4882747
4.07
1p36 microdeletion syndrome
19.05
chr1:1000000‐3000000
Yes
Yes
Yes
Yes

A0109
dup
chr17:16429920‐20667174
4.24
Smith‐Magenis syndrome
18.88
chr17:16000000‐19000000
Yes
Yes
Yes
Yes

A0142
del
chr22:17900000‐22200000
4.3
DiGeorge syndrome/22q11.2 deletion syndrome
11.83
chr22:170000000‐22000000
No
Yes
Yes
Yes

A1012
del
chr1:749625‐5619192
4.87
1p36.33‐36.32 Deletion
14.72
chr1:0‐5000000
Yes
Yes
Yes
Yes

A0844
del
chr21:41935392‐46880878
4.95

10.42
chr21:41000000‐47000000
No
No
Yes
Yes

A0901
dup
chrY:17801068‐22916805
5.12
AZEb
NA
chrY:20000000‐23000000
Yes
Yes
Yes
Yes

A0433
dup
chr15:23076361‐28436403
5.36
15q11.2 deletion syndrome
20.75
chr15:23000000‐27000000
Yes
Yes
Yes
Yes

A0899
del
chr6:1.65E+08‐1.71E+08
5.37
microdeletion 6q27 anosmia
8.93
chr6:164000000‐171000000
No
Yes
Yes
Yes

A0947
dup
chr15:20627802‐26109998
5.48
Prader‐Willi syndrome (type 2)
14.76
chr15:20000000‐27000000
Yes
Yes
Yes
Yes

A0133
del
chr14:1.01E+08‐1.07E+08
6.06

2.01
‐‐
No
No
No
No

A0001
dup
chr19:51529057‐59092570
7.56

19.05
chr19:50000000‐57000000
Yes
Yes
Yes
Yes

A0202
dup
chr22:17096855‐25153910
8.06
DiGeorge syndrome/22q11.2 deletion syndrome
24.87
chr22:17000000‐23000000
Yes
Yes
Yes
Yes

A0894
dup
chr3:1.90E+08‐1.99E+08
9.13
3q29 microdeletion syndrome
28.29
chr3:190000000‐199000000
Yes
Yes
Yes
Yes

A0786
del
chr4:61552‐9237101
9.18
Wolf‐Hirschhorn syndrome
22.78
chr4:0‐9000000
Yes
Yes
Yes
Yes

A0107
dup
chrY:12571053‐22916805
10.35
AZEa
23.31
chrY:5000000‐8000000
Yes
Yes
Yes
Yes

A0107
del
chrY:1091‐10379571
10.38
sex‐determining region Y/SRY
23.31
chrY:5000000‐8000000
Yes
Yes
Yes
Yes

A0011
del
chr4:61660‐10975146
10.91
Wolf‐Hirschhorn syndrome
20.89
chr4:0‐12000000
Yes
Yes
Yes
Yes

A0256
dup
chr12:45001‐11278012
11.23
12p13.33 microdeletion syndrome
19.27
chr12:0‐13000000
Yes
Yes
Yes
Yes

A0578
del
chr18:10001‐11497100
11.49
18p deletion syndrome
14.16
chr18:3000000‐11000000
Yes
Yes
Yes
Yes

A0676
del
chr4:1.78E+08‐1.91E+08
12.6
ASD:Small omphalocele. anteriorly placed anus, clef tpalate
8.54
chr4:180000000‐189000000
Yes
Yes
Yes
Yes

A0248
del
chr4:75392078‐88436655
13.04
4q21 deletion syndrome
25.90
chr4:75000000‐92000000
Yes
Yes
Yes
Yes

A0577
dup
chr18:10001‐13279511
13.27
18p deletion syndrome
11.13
chr18:0‐8000000
Yes
Yes
Yes
Yes

A0856
del
chr13:1.01E+08‐1.14E+08
13.29

17.84
chr13:101000000‐113000000
Yes
Yes
Yes
Yes

A0322
dup
chr17:87009‐13501809
13.41
17p13.1 deletion syndrome
NA
chr17:0‐13000000
Yes
Yes
Yes
Yes

A0359
del
chr18:142096‐13885315
13.74
18p deletion syndrome
15.81
chr18:0‐13000000
Yes
Yes
Yes
Yes

A0652
dup
chr13:99103482‐1.13E+08
14.32

22.09
chr13:50000000‐95000000
Yes
Yes
Yes
Yes

A1092
dup
chr18:4316‐14918854
14.91
18p11.32‐p11.31 duplication
12.48
chr18:0‐15000000
Yes
Yes
Yes
Yes

A0580
del
chr15:83800036‐99660791
15.86
15q25 deletion syndrome
8.68
chr15:69000000‐100000000
Yes
Yes
Yes
Yes

A0310
dup
chr11:1.17E+08‐1.35E+08
18.14
Jacobsen syndrome
14.74
chr11:116000000‐131000000
Yes
Yes
Yes
Yes

A0953
del
chr4:61552‐18242617
18.18
4p16.3‐16.1 Deletion
9.45
chr4:0‐18000000
Yes
Yes
Yes
Yes

A0676
dup
chr3:93949‐19556862
19.46
3p syndrome
8.54
chr3:0‐20000000
Yes
Yes
Yes
Yes

A0856
dup
chr5:204737‐20929211
20.72
Cri du Chat syndrome (5p deletion)
17.84
chr5:7000000‐21000000
Yes
Yes
Yes
Yes

A0686
dup
chr10:1.14E+08‐1.35E+08
21.26
10q26 deletion syndrome
NA
chr10:113000000‐133000000
Yes
Yes
Yes
Yes

A0224
dup
chr7:1.37E+08‐1.59E+08
22.01

17.61
chr7:135000000‐157000000
Yes
Yes
Yes
Yes

A1073
dup
chr2:161989273‐185124629
23.14
2q31.1 duplication syndrome
9.78
chr2:163000000‐18000000
Yes
Yes
Yes
Yes

A0202
dup
chr7:1.36E+08‐1.59E+08
23.16

24.87
chr7:136000000‐157000000
Yes
Yes
Yes
Yes

A0359
del
chr18:53764221‐77982126
24.22
18q deletion syndrome
15.81
chr18:53000000‐76000000
Yes
Yes
Yes
Yes

A0302
dup
chr13:90000001‐1.15E+08
25.17

19.03
chr13:91000000‐113000000
Yes
Yes
Yes
Yes

A0644
del
chr13:37474970‐65394654
27.92
13q14 deletion syndrome
14.70
chr13:40000000‐66000000
Yes
Yes
Yes
Yes

A0224
dup
chr13:80744674‐1.13E+08
32.68

17.61
chr13:82000000‐113000000
Yes
Yes
Yes
Yes

A0894
del
chr13:74724554‐1.14E+08
39.35

28.29
chr13:81000000‐113000000
Yes
Yes
Yes
Yes

A0256
dup
chr14:20472548‐64879219
44.41
14q11‐q22 deletion syndrome
19.27
chr14:20000000‐62000000
Yes
Yes
Yes
Yes

A0519
dup
chr18:33381218‐783231 17
44.94
18q deletion syndrome
9.57
chr18:33000000‐76000000
Yes
Yes
Yes
Yes

A0093
dup
chr5:0‐46100000
46.1
Cri du Chat syndrome (5p deletion)
NA
chr5:1000000‐45000000
Yes
Yes
Yes
Yes

A0403
dup
chr21:1‐48129895
48.13
Early‐onset Alzheimer disease with cerebral amyloid
10.58
chr21:15000000‐46000000
Yes
Yes
Yes
Yes

angiopathy

A0322
del
chrX:61529‐48771280
48.71
Xp11.22‐p11.23 microduplication
NA
chrX:3000000‐48000000
Yes
Yes
Yes
Yes

A0899
dup
chr2:1.90E+08‐2.43E+08
53.11
2q33.1 deletion syndrome
8.93
chr2:188000000‐241000000
Yes
Yes
Yes
Yes

A0971
del
chrX:90374470‐155190083
64.82
Xq26‐28 Deletion
11.03
chrX:93000000‐150000000
Yes
Yes
Yes
Yes

A0632
dup
chr9:261257‐84260002
84
9p deletion syndrome
14.90
chr9:0‐37000000
Yes
Yes
Yes
Yes

Note: Syndrome is associated with either deletion or duplication in the region but may not match the observed DNA abnormality

Dub = Duplication; Del = Deletion;; FC = Fetal DNA Concentraction