Applying high‐throughput sequencing to identify and evaluate foetal chromosomal deletion and duplication

Abstract The present study aimed to estimate the clinical performance of non‐invasive prenatal testing (NIPT) based on high‐throughput sequencing method for the detection of foetal chromosomal deletions and duplications. A total of 6348 pregnant women receiving NIPT using high‐throughput sequencing method were included in our study. They all conceived naturally, without twins, triplets or multiple births. Individuals showing abnormalities in NIPT received invasive ultrasound‐guided amniocentesis for chromosomal karyotype and microarray analysis at 18‐24 weeks of pregnancy. Detection results of foetal chromosomal deletions and duplications were compared between high‐throughput sequencing method and chromosomal karyotype and microarray analysis. Thirty‐eight individuals were identified to show 51 chromosomal deletions/duplications via high‐throughput sequencing method. In subsequent chromosomal karyotype and microarray analysis, 34 subchromosomal deletions/duplications were identified in 26 pregnant women. The observed deletions and duplications ranged from 1.05 to 17.98 Mb. Detection accuracy for these deletions and duplications was 66.7%. Twenty‐one deletions and duplications were found to be correlated with the known abnormalities. NIPT based on high‐throughput sequencing technique is able to identify foetal chromosomal deletions and duplications, but its sensitivity and specificity were not explored. Further progress should be made to reduce false‐positive results.

for newborns. 6 Conventional prenatal testing techniques include karyotyping, comparative genomic hybridization (CGH), hybridization and array-based technologies. 7 These conventional screening methods require foetal DNA samples through invasive approaches, like amniocentesis, which may increase the risk of miscarriage and infection. 8 Moreover, conventional testing techniques could only detect deleted and duplicated fragments of more than 10 Mb, and abnormalities with microdeletion and microduplication may be undetectable. 9 Therefore, non-invasive prenatal genetic screening methods with high accuracy are in urgent need.
In 1997, Lo et al reported the presence of cell-free foetal DNA (cffDNA) in maternal plasma that allows the application of non-invasive prenatal testing (NIPT) in clinical practice. 10 cffDNA in maternal plasma mainly derives from placenta, especially from the outer cytotrophoblastic layer. 11 cffDNA shows linear correlation with chromosomal abnormalities in foetuses and is considered as the optimal proxy in NIPT. 12 NIPT based on high-throughput sequencing technique can effectively detect large-scale genetic mutations in a short time, with high accuracy. 13 Compared to conventional prenatal testing, high-throughput NIPT has multiple advantages. First, it causes no risk of pregnancy loss thanks to its non-invasive procedures. 14 Second, it has been reported that the detection rate of high-throughput sequencing for trisomy 21, trisomy 18 and trisomy 13 may be up to 79%. 15 Third, the technique is suitable for varied gestational ages, even after 23 weeks of pregnancy. In addition, the operational process is simple and automated. However, the technique is not suitable in the detection for multiple births. Moreover, its detection rate for deletions and duplications less than 10 Mb is unsatisfactory. The performance of NIPT using high-throughput sequencing technique for chromosomal deletions and duplications remained controversial.
In this study, we estimated the performance of NIPT based on high-throughput sequencing for foetal deletions and duplications.

| Study subjects
A total of 6348 eligible pregnant women were retrospectively recruited in the current study from May 2015 to January 2019. The all conceived naturally, without twins, triplets or multiple births. The included pregnant women received NIPT which was performed via high-throughput sequencing method, regardless of whether they experienced any Down syndrome examinations. Moreover, the age of the eligible subjects was over 18 years, with a pregnancy of more than 12 weeks. The pretest ultrasound scan was performed for each subject to confirm the number of foetuses and gestational age. In addition, women who had a foetus with major structural abnormalities were excluded from this study. Written informed consent was signed by each woman before inclusion. The current investigation was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University.

| Blood sample and DNA extraction
Five millilitres of peripheral blood from each pregnant woman was collected into a cell-free DNA tube (Streck, Omaha, NE, USA). Then, cell-free plasma was isolated from the obtained blood samples via a two-step centrifugation method within 4 hours after collection.
In brief, blood samples were first centrifuged at 1600 g for 10 minutes at 4°C, and then supernatant was transferred into a new tube and centrifuged for additional 10 minutes at 1600 g under 4°C. Final plasma supernatant was transferred to a cell-free DNA tube and then stored at −20°C for DNA extraction. Each plasma sample was thawed only once.
Cell-free DNA was extracted from plasma specimens using a Dynabeads ® Viral NA DNA purification kit (Dynal, Grand Island, NY, USA), and experiment procedures were performed based on the instruction of manufacturer. DNA samples were stored at −80°C.

| DNA library construction
Firstly, cell-free DNA samples were quantified by Qubit 3.0 fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, USA). No less than 10 ng DNA sample was collected from each woman, and DNA concentration was over 1.7 ng/mL. Qualified DNA samples were adopted for PCR amplification, and reaction procedures were as follows: at 99°C for 2 minutes, and 22 cycles of 99°C for 15 seconds and 60°C for 4 minutes. After primer digestion, amplification products were ligated with adaptors and purified by Agecoure AMPure

| Sequencing template preparation and enrichment
Sequencing templates were prepared and enriched according to the standard procedures recommended by Life Technology Company.
The template was prepared through emulsion PCR, which was performed using Ion temple preparation kit (Life Technologies). The reaction was carried out in a volume of 1 mL mixture including 582 μL nuclease-free water, 200 μL 5× PCR reagent mix, 100 μL 10× PCR enzyme mix, 100 μL Ion Sphere particles (ISPs) and 18 μL diluted library template. The mixture was shaken and centrifuged, and Ultra-Turrax tube drive (Life Technologies) was adopted for emulsion. Then, the mixed emulsion was transferred to 96-well plate and amplified on an ABI 2720 thermocycler (Life Technologies).
After amplification, ISP was recovered using Ion Xpress template kit (Life Technologies) following the instruction of manufacturer. Q ubit 3.0 fluorometer and Ion Sphere quality control kit were applied for particle quantification. The optimal positive ISPs for enrichment were 4%-50%. Finally, ISP enrichment was performed using Ion Xpress template kit, Ion sequencing kit and DynaBeads MyOne streptavidin C1 beads (Life Technologies), and experiments were carried out according to the guidance of manufacturer.

| Ion torrent proton sequencing
The prepared sequencing template was annealed on PCR amplification thermocycler, and the parameters were set as follows: 95°C for 2 minutes, 37°C for 2 minutes and 25°C for storage. The annealed template was loaded and run with 200-bp single-end run configuration based on the manufacturer's instruction (Ion sequencing kit user guide, version 2.0).

| Bioinformatics analysis
Sequencing results were collected and analysed using VariantCaller software (Life Technologies). In comparison with human genome reference sequence (GRCh37 Sequences), chromosomal deletion and duplication were analysed. Reference genome was divided into 300 000 sliding windows which contained the same number of reads, and relative number of reads was defined as the ratio of the number of reads in an equal window to the average number of reads. The least square method was adopted to analyse linear relationship between GC content and relative number of reads. The types of foetal chromosome abnormality were predicted through dynamic threshold method and quadratic element segmentation algorithm.

| Chromosome karyotype analysis and microarray analysis
Individuals who showed abnormalities in NIPT received ultrasoundguided amniocentesis at 18-24 weeks of pregnancy after informed consent was signed by pregnant women and their families. Amniotic fluid samples were collected from the patients for conventional G-banded cytogenetic assays and microarray analysis. Amniocytes were isolated and cultured using BIO-AMF™-2 medium (Biological Industries, Kibbutz Beit-Haemek, Israel) and Chang Medium ® D (Irvine Scientific, Santa Ana, CA, USA) at 37°C with 5% CO 2 for 6-7 days.
The cells at metakinesis were harvested to prepare slides according to the statements in published article. 16 Then, G-band staining was

| Overall results of NIPT
The results of NIPT based on high-throughput sequencing method were available for all of the included patients. As displayed in Figure 1,  (Figure 2). In addition, 17 abnormalities in 12 cases were misdiagnosed in NIPT, and the false-positive rate was 33.3%. Detailed descriptions for false-positive results were listed in Table 3. The size of these deletions/duplications ranged from 0.50 to 4.37 Mb, and the major of them were less than 1.5 Mb.  Besides, limited by current genome function, a certain proportion of NIPT results were inconclusive. 28 Additionally, in our study only individuals showing abnormalities in NIPT received chromosome karyotype analysis and microarray analysis, considering invasive procedures of amniocentesis and reported low negative rate of NIPT. Individuals who might be misdiagnosed in NIPT did not experience chromosome karyotype analysis, and false-negative rate of NIPT was not calculated.

| D ISCUSS I ON
Therefore, much more progress should be made to translate NIPT results in clinical practice.
In conclusion, NIPT based on high-throughput sequencing technique is able to identify foetal chromosomal deletions and duplications. However, due to the relatively low foetal DNA concentration, small abnormal fragments and limited sequencing depth, its false-positive rate may be high.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.