Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.
Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60−80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2−4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.
Choosing good-quality viable embryos with normal chromosome copy numbers (euploid) for transfer in assisted reproduction helps to improve pregnancy outcomes. Traditionally, the well-established morphological grading system is widely used for embryo evaluation due to its easy availability and non-invasive nature. However, it has been shown that morphological assessment can only provide limited information on embryo quality1 and implantation potential2. One fundamental reason is its inability in evaluating the chromosomal composition of the embryos.
Chromosomal aneuploidy (abnormal copy number of chromosomes) is one of the main causes leading to embryonic development arrest, implantation failure or pregnancy loss. The occurrence of aneuploidy has been well documented in human embryos, accounting for 60%−70% in cleavage-stage embryos3,4 and 50%−60% in blastocysts5. This, to some extent, has contributed to the bottleneck in improving the pregnancy rate of in-vitro fertilization (IVF) treatment, which has maintained at around 35%−40%6,7. Therefore, selecting euploid embryos for transfer is believed to be beneficial for improving pregnancy outcomes. To this end, preimplantation genetic testing for aneuploidy (PGT-A) has been further developed to investigate embryo viability using genetic approaches. There are increasing numbers of randomized controlled trials and cohort studies supporting the crucial role of PGT-A. It has been proved that the application of PGT-A decreases the miscarriage rate and increases clinical pregnancy rate and implantation rate8, ongoing pregnancy rate and live birth rate9.
Historically, different methods have been applied in PGT-A, such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), array-CGH, and single nucleotide polymorphism (SNP)-microarray. Previous studies have indicated that PGT-A for cleavage-stage embryos by FISH yields results that are poorly consistent with those obtained by comprehensive chromosomal screening (CCS) of corresponding blastocysts using 59273array-CGH or SNP-microarray5927310. These discrepancies can be attributed to chromosomal mosaicism, FISH technical artifacts, or embryonic self-correction of chromosomal segregation errors during development11. It has been widely recognized that using blastocyst trophectoderm (TE) biopsies for array-based PGT-A such as array-CGH or SNP-microarray is effective for identifying the chromosomal imbalance in embryos10,12. Recently, single-cell next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A13,14,15, which make it a promising alternative to currently available methods.
Here, we present a fast, robust, and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in human embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, using a single-cell WGA kit, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system.
Through detecting the H+ ions that are released from each deoxyribonucleoside triphosphate incorporation during DNA strand synthesis, the system transfers the chemical signals (pH change) captured by the semiconductor elements to direct digital data, which are further interpreted into DNA sequence information. Eliminating the requirement for expensive optical detection and complex sequencing reactions, this simple sequencing chemistry reduces total reagent cost and shortens the sequencing running time into 2−4 hours16. More importantly, based on the manufacturer’s performance specifications, the semiconductor sequencing platform can generate up to 15 GB sequencing data (depends on the quality of library) per run, which is significantly higher than some of the other sequencers producing only around 3−4 GB data (with 2 x 75 bp read length)17. In clinical applications of PGT-A, this platform can achieve 24 samples per chip generating up to 80 million reads17 and at least one million unique reads of each sample. The read depth can ensure that each sample has at least 0.05x whole genome coverage. The above advantages of this platform make it an ideal screening method and thus, facilitate its wide applications in PGT-A18.
Ethical approval was granted by the Joint Chinese University of Hong Kong―New Territories East Cluster Clinical Research Ethics Committee (Reference Number: 2010.432). Research license was approved by the Council on Human Reproductive Technology of Hong Kong (Number R3004).
1. Whole genome amplification
2. Quality control of the WGA products
3. Fragmentation of WGA products
4. Library construction
5. Quality control and dilution of the DNA library
6. Sequencing
Based on this modified protocol, the semiconductor sequencing platform was for the first time, applied for PGT-A. We tested on biopsies from both cleavage-stage blastomeres and blastocyst-stage embryos. It is suggested that the biopsied cells undergo WGA as soon as possible to prevent any degradation of DNA. A previous study compared the performance of different WGA methods and indicated that the method we described here had the best uniformity at the bin size of 100 KB20. Considering the performance of both uniformity and median absolute pairwise difference (MAPD)21, this WGA method was chosen for PGT-A using the semiconductor sequencer. Through a retrospective statistical analysis on 186 cleavage stage and 1135 blastocyst stage embryos, we observed that the WGA success rates were 95.4% in blastomere samples and 96.9% in blastocyst samples (Figure 1). The purification step before library construction as a size selection procedure was crucial for sequencing quality by capturing large DNA fragments. Additionally, it facilitated the input amount of 300 ng for library construction. The enzymatic fragmentation method enabled an efficient shearing of WGA products into approximately 160 bp.
Data analysis was conducted using the Euclidean distance and circular binary segmentation (EDCBS) analysis system. In-house validation was performed to evaluate the robustness of this bioinformatic algorithm. We established a reference database exclusive for PGT-A through sequencing 379 WGA products from 66 cell lines with known karyotypes by analyzing a bin size of 100 KB. From this database, a reference range was delineated as the threshold for copy number variant (CNV) calling and 10 MB was set as the cutoff for the detection level. Both sensitivity and specificity reached over 99% at this threshold (Table 1). In the application for PGT-A on embryo biopsies, the window size was set to 400 KB with a sliding window approach to reach enough reads. Quality control (QC) of each sample was determined by unique reads, MAPD and standard deviation of copy number variant (CNV∙SD). Sample beyond one of the three indexes was defined as QC failure (Figure 2C). Interpretation of chromosome scatter plots (Figure 2A,B,D) was conducted by qualified geneticists following a workflow by comparing the identified CNV to DECIPHER, DGV, or ClinGen databases. Individual discrepancies were controlled by an expert curation procedure. Chromosomal abnormalities were grouped into aneuploidy and mosaicism in blastocyst samples. A copy number gain or loss within the range of 30%−70% was classified as carrying mosaic chromosomal composition; otherwise, the result would be interpreted as either euploidy or aneuploidy. In this study, the euploid rates were 45.2% in blastomeres and 52.3% in blastocysts, which echoed to published data22,23.
Figure 1: Demographic statistics of 1321 embryo biopsies tested by this method. (A) Data from 186 cleavage-stage embryos. (B) Data from 1135 blastocyst-stage embryos. WGA success rates are over 95% in both types of specimens. Sequencing quality control failure rates are 3.4% in the cleavage-stage group and only 1.9% in the blastocyst-stage group. Please click here to view a larger version of this figure.
Figure 2: Representative results of PGT-A clinical application of embryo for the 23 pairs of chromosomes. Representative results of (A) euploidy; (B) aneuploidy (seq[GRCh37] (2)x3, (21)x3); (C) sequencing QC failed sample due to CNV∙SD at 0.6571 (acceptance ≤ 0.4); (D) segmental mosaic deletion (55%) of 4p16.3p15.1 (29.50 MB). Please click here to view a larger version of this figure.
CNV threshold value | ||||||
(-0.23, 0.20) | (-0.32, 0.26) | (-0.41, 0.32) | (-0.51, 0.37) | (-0.62, 0.43) | (-0.73, 0.48) | |
Sensitivity | 100.00% | 100.00% | 100.00% | 100.00% | 98.30% | 96.02% |
Specificity | 72.41% | 81.03% | 91.38% | 99.10% | 99.54% | 100.00% |
Table 1: Sensitivity and specificity between different Log R ratios by the semiconductor sequencer. A total of 240 samples with known euploid karyotype results were tested by this method and called at different Log R ratios.
Different from other sequencing chemistries, the sequencer described here uses semiconductor for the detection of nucleotides. The chip itself is an electronic device that detects hydrogen ions by polymerase-driven base incorporation17, which enables 2−4 h sequencing time of the Proton program. Besides, the chip is a microwell chip that allows the localization of one target molecule, which is different from the flow cell sequencing chemistry by other sequencer providers. This protocol is a modified protocol optimized for the application of PGT-A. The optimization includes fragmentation of amplified DNA by the enzymatic method instead of sonication to reduce the chance of contamination as well as the size-selection and the PCR system for better performance with lower costs. Additionally, in the clinical setting, we used a validated in-house pipeline for variant calling.
There are critical steps to be aware of during the practice. Ethanol for purification needs to be freshly prepared before the experiment, as a high concentration of ethanol would cause insufficient wash of contaminated DNA while a low concentration would cause loss of target DNA. The fluorometer has to be calibrated by the positive control with a known concentration to ensure the accuracy. Besides, an adequate loading of the template is crucial for sequencing. Bubbles of the right size help to push the template sphere to fall into microwells, but too large bubbles may cause inadequate loading. Users can modify the number of samples (not necessarily 24) to be sequenced for each run. There are 96 indexes designed for this chip. But sequencing read depth will decrease with increasing samples per chip. One of the most common problems is low library concentration, which can be attributed to a low or poor-quality DNA output from purification due to residual ethanol or magnetic beads, or the cracking of beads as previously mentioned. A suboptimal DNA output may also result from low efficiency of PCR, which can be corrected by cautious preparation of the master mix with accurate sample inputs and temperature check of the thermal cyclers. For sequencing QC-failed samples, such as those with high CNV∙SD values (Figure 2C), it is recommended to run the samples on a bioanalyzer for size distribution analysis.
One of the limitations of this method is its higher false single-nucleotide calling rate compared to other platforms. The error rate is 1% compared to only 0.1% indel false positive rate by other sequencers17. However, this is not a determining factor for CNV calling or PGT-A analysis. Compared to other platforms, application of the emulsion system minimizes operative discrepancies and pipetting errors, increasing the library quality. This is a significant advantage of our method compared to other platforms because the dsDNA concentration is very low and an accurate quantification is required for the following library pooling. Our method introduced the calibration of fluorometer quantification using a standard positive control to control detection error.
As a high throughput platform with short turnaround time, the semiconductor sequencer is ideal for PGT-A and can be widely applied to IVF patients with PGT-A clinical indications such as advanced maternal age24. Deleye et al. conducted parallel sequencing of human blastocysts to compare the performance of the semiconductor sequencer with other sequencers25. Moreover, this PGT-A kit has obtained the “special approval procedure on innovative medical devices” from the China Food and Drug Administration and has been clinically used for thousands of embryos. In terms of cost, the price of this platform is half the price per giga bases compared to another commonly used platform in the PGT-A market. The trophectoderm cells can reliably represent the genetic constitution of the embryo26. This method can potentially be developed for PGT for monogenic/single gene diseases (PGT-M) as Treff et al. have demonstrated27. In their model, they facilitated 300 MB to 1 GB throughput on PGT-M of 16 embryo biopsies and compared the results to two conventional PGT-M methods. By capturing and loading 16 samples, their method reached at least 100x read depth of the targeted region and resulted in 100% reliability and reproducibility27. The throughput of the semiconductor sequencer by our protocol can reach 15 GB and there are 96 barcodes designed; therefore, if applied to targeted PGT-M, a considerable number of embryo biopsies can be sequenced by one chip at a high read depth.
The authors have nothing to disclose.
This study was supported by the General Research Fund (Ref No. 14162417) from Hong Kong, the National Natural Science Foundation of China (Ref No. 81860272), the Major Research Plan of the Provincial Science and Technology Foundation of Guangxi (Ref No. AB16380219), and the China Postdoctoral Science Foundation Grant (Ref No. 2018M630993) from China.
PCR tubes, 0.2 mL | Axygen | PCR-02D-C | |
UltraPure 0.5M EDTA, pH 8.0 | ThermoFisher | 15575020 | |
PBS, pH 7.4, Ca2+ and Mg2+ free | ThermoFisher | 10010023 | |
1.0 M NaOH (1.0N) solution | SIGMA-ALDRICH | S2567 | For Melt-off solution. Molecular grade |
Eppendorf LoBind Tubes, 1.5 mL | Fisher Scientific | 13-698-791 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
ELGA PURELAB Flex 3 Water Purification System or Equivalent 18 MΩ water system |
ThermoFisher | 4474524 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
PicoPLEX WGA Amplification buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
PicoPLEX WGA Amplification enzyme | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Ion OneTouch Amplification Plate | In kit: Ion OneTouch 2 Supplies (Part No. A26367). Extended kit component in Sheet 5 | ||
Ion PI Annealing Buffer | |||
MyOne Beads Capture Solution | |||
Agilent 2100 Bioanalyzer instrument | Agilent | G2939AA | |
Ion OneTouch Breaking Solution (black cap) | In kit: Ion PI Hi‑Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5 | ||
Dynabeads MyOne Streptavidin C1 | ThermoFisher | 65001 | |
PicoPLEX WGA Cell extraction buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-00. |
PicoPLEX WGA Cell extraction enzyme | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Ion PI Chip Kit v3 | ThermoFisher | A26771 | |
Ion Chip Minifuge, 230 V | ThermoFisher | 4479673 | |
Ion PI dATP | ThermoFisher | A26772 | |
Ion PI dCTP | ThermoFisher | A26772 | |
Ion PI dGTP | ThermoFisher | A26772 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
ThermoQ–Temperature Dry Bath | TAMAR | HB-T2-A | |
NEBNext dsDNA Fragmentase | New England Biolabs | M0348L | |
NEBNext dsDNA Fragmentase | New England Biolabs | M0348L | |
Ion PI dTTP | ThermoFisher | A26772 | |
Ion OneTouch 2 Instrument | ThermoFisher | INS1005527 | ThermoFisher Catalog number: 4474778. |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
Ion One Touch ES | ThermoFisher | 8441-22 | ThermoFisher Catalog number: 4469495. Extended kit component in Sheet 5 |
Ethanol | SIGMA-ALDRICH | 51976 | This can be replaced by any brand's molecular grade absolute ethanol. |
PicoPLEX WGA Extraction enzyme dilution buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-01. |
PicoPLEX WGA Extraction enzyme dilution buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-02. |
Qubit 3.0 Fluorometer | ThermoFisher | Q33216 | This model has been replaced by Qubit 4 Fluorometer, Catalog number: Q33226. |
Qubit ds DNA HS Assay kit | ThermoFisher | M2002-02 | |
Qubit Assay Tubes | ThermoFisher | Q32856 | |
Ion PI Foaming Solution | ThermoFisher | A26772 | |
Index for barcoding of libraries | BaseCare | this is a in-house prepared index. Users can buy commercial product from ThermoFisher Ion Xpress Barcode Adapters Kits (Cat. No. 4474517) | |
Ion PI Loading Buffer | ThermoFisher | A26772 | |
Solid(TM) Buffer Kit-1X Low TE Buffer | ThermoFisher | 4389764 | |
Agencour AMPure XP Kit | Beckman Coulter | A63880 | |
DynaMag-2 magnet (magnetic rack) | ThermoFisher | 12321D | |
Ion PI Master Mix PCR buffer | |||
Sorvall Legend Micro 17 Microcentrifuge | Micro 17 | 75002430 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
Nuclease-free water | ThermoFisher | AM9922 | This can be replaced by other brand. |
PicoPLEX WGA Nuclease-free water | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Ion OneTouch Oil bottle | Ion PI Hi‑Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5 | ||
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | Extended kit component in Sheet 3 |
double-strand DNA standard | This is a in-house prepared DNA standard for calibration of Qubit before quantification of library. | ||
PicoPLEX WGA Preamplification buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
PicoPLEX WGA Preamplification enzyme | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Library Amplification Primer Mix | ThermoFisher | 4471252 | Extended kit component in Sheet 3 |
Ion OneTouch Reaction Filter | Extended kit component in Sheet 5 | ||
Recovery Router | Extended kit component in Sheet 5 | ||
Recovery Tubes | Extended kit component in Sheet 5 | ||
ISP Resuspension Solution | |||
Ion Proton | ThermoFisher | DA8600 | This model is imported by Da An Gene Co.,LTD. of Sun Yat-Sen University from ThermoFisher and has been certified by China Food and Drug Administration for clinical application. The catalog number in ThermoFisher is 4476610. |
Ion PI Hi‑Q Sequencing Polymerase | ThermoFisher | A26772 | |
Ion PI Sequencing Primer | |||
server for sequencer | Lenovo | T260 | |
Ion PI Sphere Particles | |||
Platinum PCR SuperMix High Fidelity | ThermoFisher | 4471252 | |
Nalgene 25mm Syringe Filters | ThermoFisher | 724-2045 | Pore size: 0.45μm. Specifically for aqueous fluids. |
Ion PI Hi‑Q W2 Solution | ThermoFisher | A26772 | |
Ion PI 1X W3 Solution | ThermoFisher | A26772 | |
Ion OneTouch Wash Solution C1 | |||
The Ion PGM Hi‑Q View Sequencing Kit | ThermoFisher | A30044 | Extended kit component in Sheet 2 |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | Extended kit component in Sheet 3 |
Ion PI Hi-Q Sequencing 200 Kit (1 sequencing run per initialization) | ThermoFisher | A26772 | Extended kit component in Sheet 4 |
Ion PI Hi‑Q OT2 200 Kit | ThermoFisher | A26434 | Extended kit component in Sheet 5 |