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* These authors contributed equally
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 o....
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
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 KB2.......
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 m.......
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.
....Name | Company | Catalog Number | Comments |
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 |
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