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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. Prior to start, check the volume of magnetic beads (Table of Materials) to ensure that there is no less than 135 µL (with a 20% excess) for each sample. .......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
PCR tubes, 0.2 mLAxygenPCR-02D-C
UltraPure 0.5M EDTA, pH 8.0ThermoFisher15575020
PBS, pH 7.4, Ca2+ and Mg2+ freeThermoFisher10010023
1.0 M NaOH (1.0N) solutionSIGMA-ALDRICHS2567For Melt-off solution. Molecular grade
Eppendorf LoBind Tubes, 1.5 mLFisher Scientific13-698-791
Ion Plus Fragment Library KitThermoFisher4471252
ELGA PURELAB Flex 3 Water Purification System or
Equivalent 18 MΩ water system
ThermoFisher4474524
Ion Plus Fragment Library KitThermoFisher4471252
PicoPLEX WGA Amplification bufferRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
PicoPLEX WGA Amplification enzymeRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Ion OneTouch Amplification PlateIn 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 instrumentAgilentG2939AA
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 C1ThermoFisher65001
PicoPLEX WGA Cell extraction bufferRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-00.
PicoPLEX WGA Cell extraction enzymeRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Ion PI Chip Kit v3ThermoFisherA26771
Ion Chip Minifuge, 230 VThermoFisher4479673
Ion PI dATPThermoFisherA26772
Ion PI dCTPThermoFisherA26772
Ion PI dGTPThermoFisherA26772
Ion Plus Fragment Library KitThermoFisher4471252
Ion Plus Fragment Library KitThermoFisher4471252
ThermoQ–Temperature Dry BathTAMARHB-T2-A
NEBNext dsDNA FragmentaseNew England BiolabsM0348L
NEBNext dsDNA FragmentaseNew England BiolabsM0348L
Ion PI dTTPThermoFisherA26772
Ion OneTouch 2 InstrumentThermoFisherINS1005527ThermoFisher Catalog number: 4474778.
Ion Plus Fragment Library KitThermoFisher4471252
Ion One Touch ESThermoFisher8441-22ThermoFisher Catalog number: 4469495. Extended kit component in Sheet 5
EthanolSIGMA-ALDRICH51976This can be replaced by any brand's molecular grade absolute ethanol.
PicoPLEX WGA Extraction enzyme dilution bufferRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-01.
PicoPLEX WGA Extraction enzyme dilution bufferRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-02.
Qubit 3.0 FluorometerThermoFisherQ33216This model has been replaced by Qubit 4 Fluorometer, Catalog number: Q33226.
Qubit ds DNA HS Assay kitThermoFisherM2002-02
Qubit Assay TubesThermoFisherQ32856
Ion PI Foaming SolutionThermoFisherA26772
Index for barcoding of librariesBaseCarethis is a in-house prepared index. Users can buy commercial product from ThermoFisher Ion Xpress Barcode Adapters Kits (Cat. No. 4474517)
Ion PI Loading BufferThermoFisherA26772
Solid(TM) Buffer Kit-1X Low TE BufferThermoFisher4389764
Agencour AMPure XP KitBeckman CoulterA63880
DynaMag-2 magnet (magnetic rack)ThermoFisher12321D
Ion PI Master Mix PCR buffer
Sorvall Legend Micro 17 MicrocentrifugeMicro 1775002430
Ion Plus Fragment Library KitThermoFisher4471252
Nuclease-free waterThermoFisherAM9922This can be replaced by other brand.
PicoPLEX WGA Nuclease-free waterRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Ion OneTouch Oil bottleIon PI Hi‑Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5
Ion Plus Fragment Library KitThermoFisher4471252Extended kit component in Sheet 3
double-strand DNA standardThis is a in-house prepared DNA standard for calibration of Qubit before quantification of library.
PicoPLEX WGA Preamplification bufferRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
PicoPLEX WGA Preamplification enzymeRubicon GenomicsR30050This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.
Library Amplification Primer MixThermoFisher4471252Extended kit component in Sheet 3
Ion OneTouch Reaction FilterExtended kit component in Sheet 5
Recovery RouterExtended kit component in Sheet 5
Recovery TubesExtended kit component in Sheet 5
ISP Resuspension Solution
Ion ProtonThermoFisherDA8600This 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 PolymeraseThermoFisherA26772
Ion PI Sequencing Primer
server for sequencerLenovoT260
Ion PI Sphere Particles
Platinum PCR SuperMix High FidelityThermoFisher4471252
Nalgene 25mm Syringe FiltersThermoFisher724-2045Pore size: 0.45μm. Specifically for aqueous fluids.
Ion PI Hi‑Q W2 SolutionThermoFisherA26772
Ion PI 1X W3 SolutionThermoFisherA26772
Ion OneTouch Wash Solution C1
The Ion PGM Hi‑Q View Sequencing KitThermoFisherA30044Extended kit component in Sheet 2
Ion Plus Fragment Library KitThermoFisher4471252Extended kit component in Sheet 3
Ion PI Hi-Q Sequencing 200 Kit (1 sequencing run per initialization)ThermoFisherA26772Extended kit component in Sheet 4
Ion PI Hi‑Q OT2 200 KitThermoFisherA26434Extended kit component in Sheet 5

References

  1. Balaban, B., Urman, B. Effect of oocyte morphology on embryo development and implantation. Reproductive BioMedicine Online. 12 (5), 608-615 (2006).
  2. Capalbo, A., et al.

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