Name: Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy (Video) | JoVE Journal
Uploaded: 2019-08-25T13:00:00.000+00:00
Duration: 9 s
Description: Learn semiconductor sequencing for preimplantation genetic testing for aneuploidy using embryo samples. This rapid, cost-effective method delivers reliable genome sequencing with high throughput and results within five days.

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5 minutes

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.

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<li>Calhaz-Jorge, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assisted+reproductive+technology+in+Europe%2C+2013%3A+Results+generated+from+European+registers+by+ESHRE%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Assisted reproductive technology in Europe, 2013: Results generated from European registers by ESHRE.</a> Human Reproduction. 32 (10), 1957-1973 (2017).</li>
<li>Dyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((International+committee+for+monitoring+assisted+reproductive+technologies+world+report%3A+Assisted+reproductive+technology+2008%2C+2009+and+2010%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">International committee for monitoring assisted reproductive technologies world report: Assisted reproductive technology 2008, 2009 and 2010.</a> Human Reproduction. 31 (7), 1588-1609 (2008).</li>
<li>Dahdouh, E. M., Balayla, J., García-Velasco, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+chromosome+screening+improves+embryo+selection%3A+A+meta-analysis%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Comprehensive chromosome screening improves embryo selection: A meta-analysis.</a> Fertility and Sterility. 104 (6), 1503-1512 (2015).</li>
<li>Chen, M., Wei, S., Hu, J., Quan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Can+Comprehensive+Chromosome+Screening+Technology+Improve+IVF%2FICSI+Outcomes%3F+A+Meta-Analysis%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Can Comprehensive Chromosome Screening Technology Improve IVF/ICSI Outcomes? A Meta-Analysis.</a> PloS One. 10 (10), e0140779(2015).</li>
<li>Northrop, L. E., Treff, N. R., Levy, B., Scott, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SNP+microarray-based+24+chromosome+aneuploidy+screening+demonstrates+that+cleavage-stage+FISH+poorly+predicts+aneuploidy+in+embryos+that+develop+to+morphologically+normal+blastocysts%5BTitle%5D)+AND+%22Molecular+Human+Reproduction%22%5BJournal%5D)">SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.</a> Molecular Human Reproduction. 16 (8), 590-600 (2010).</li>
<li>Barbash-Hazan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preimplantation+aneuploid+embryos+undergo+self-correction+in+correlation+with+their+developmental+potential%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential.</a> Fertility and Sterility. 92 (3), 890-896 (2009).</li>
<li>Fragouli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+cytogenetic+analysis+of+the+human+blastocyst+stage%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Comprehensive cytogenetic analysis of the human blastocyst stage.</a> Fertility and Sterility. 90 (11), S36(2008).</li>
<li>Fiorentino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+a+next-generation+sequencing-based+protocol+for+24-chromosome+aneuploidy+screening+of+embryos%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos.</a> Fertility and Sterility. 101 (5), 1375-1382 (2014).</li>
<li>Fiorentino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+next-generation+sequencing+technology+for+comprehensive+aneuploidy+screening+of+blastocysts+in+clinical+preimplantation+genetic+screening+cycles%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles.</a> Human Reproduction. 29 (12), 2802-2813 (2014).</li>
<li>Wells, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+utilisation+of+a+rapid+low-pass+whole+genome+sequencing+technique+for+the+diagnosis+of+aneuploidy+in+human+embryos+prior+to+implantation%5BTitle%5D)+AND+%22Journal+of+Medical+Genetics%22%5BJournal%5D)">Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation.</a> Journal of Medical Genetics. 51 (8), 553-562 (2014).</li>
<li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+tale+of+three+next+generation+sequencingplatforms%3A+comparison+of+Ion+Torrent%2C+PacificBiosciences+and+Illumina+MiSeq+sequencers%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">A tale of three next generation sequencingplatforms: comparison of Ion Torrent, PacificBiosciences and Illumina MiSeq sequencers.</a> BMC Genomics. 13 (1), 1-13 (2012).</li>
<li>Goodwin, S., McPherson, J. D., McCombie, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coming+of+age%3A+ten+years+of+next-generation+sequencing+technologies%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Coming of age: ten years of next-generation sequencing technologies.</a> Nature Reviews Genetics. 17 (6), 333-351 (2016).</li>
<li>Bono, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validation+of+a+semiconductor+next-generation+sequencing-based+protocol+for+preimplantation+genetic+diagnosis+of+reciprocal+translocations%5BTitle%5D)+AND+%22Prenatal+Diagnosis%22%5BJournal%5D)">Validation of a semiconductor next-generation sequencing-based protocol for preimplantation genetic diagnosis of reciprocal translocations.</a> Prenatal Diagnosis. 35 (10), 938-944 (2015).</li>
<li>Harton, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ESHRE+PGD+Consortium%2FEmbryology+Special+Interest+Groupbest+practice+guidelines+for+polar+body+and+embryo+biopsy+for+preimplantation+genetic+diagnosis%2Fscreening+%28PGD%2FPGS%29%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">ESHRE PGD Consortium/Embryology Special Interest Groupbest practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).</a> Human Reproduction. 26 (1), 41-46 (2011).</li>
<li>Liu, W. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+performance+of+MALBAC+and+MDA+methods+in+the+identification+of+concurrent+mutations+and+aneuploidy+screening+to+diagnose+beta-thalassaemia+disorders+at+the+single-+and+multiple-cell+levels%5BTitle%5D)+AND+%22Journal+of+Clinical+Laboratory+Analysis%22%5BJournal%5D)">The performance of MALBAC and MDA methods in the identification of concurrent mutations and aneuploidy screening to diagnose beta-thalassaemia disorders at the single- and multiple-cell levels.</a> Journal of Clinical Laboratory Analysis. 32 (2), 1-8 (2018).</li>
<li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+comparison+of+the+performance+of+four+whole+genome+amplification+kits+on+ion+proton+platform+in+copy+number+variation+detection%5BTitle%5D)+AND+%22Bioscience+Reports%22%5BJournal%5D)">The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection.</a> Bioscience Reports. 37 (4), BSR20170252(2017).</li>
<li>Munné, S., Grifo, J., Wells, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosaicism%3A+%E2%80%9Csurvival+of+the+fittest%E2%80%9D+versus+%E2%80%9Cno+embryo+left+behind%E2%80%9D%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Mosaicism: “survival of the fittest” versus “no embryo left behind”.</a> Fertility and Sterility. 105 (5), 1146-1149 (2016).</li>
<li>Del Carmen Nogales, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+of+chromosome+abnormality+affects+embryo+morphology+dynamics%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Type of chromosome abnormality affects embryo morphology dynamics.</a> Fertility and Sterility. 107 (1), 229-235 (2017).</li>
<li>Harton, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ESHRE+PGD+consortium+best+practice+guidelines+for+amplification-based+PGD%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">ESHRE PGD consortium best practice guidelines for amplification-based PGD.</a> Human Reproduction. 26 (1), 33-40 (2011).</li>
<li>Deleye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shallow+whole+genome+sequencing+is+well+suited+for+the+detection+of+chromosomal+aberrations+in+human+blastocysts%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Shallow whole genome sequencing is well suited for the detection of chromosomal aberrations in human blastocysts.</a> Fertility and Sterility. 104 (5), 1276-1285 (2015).</li>
<li>Bielanska, M., Tan, S. L., Ao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+mosaicism+throughout+human+preimplantation+development+in+vitro%3A+incidence%2C+type%2C+and+relevance+to+embryo+outcome%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome.</a> Human Reproduction. 17 (2), Oxford, England. 413-419 (2002).</li>
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</ol>

The authors have nothing to disclose.

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&#8722;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...

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%&#8722;70% in cleavage-stage embryos3,4 and 50%&#8722;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%&#8722;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&#8722;4 hours16. More importantly, based on the manufacturer&#8217;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&#8722;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.

<table><tbody><tr><td>PCR tubes, 0.2 mL</td><td>Axygen</td><td>PCR-02D-C</td><td></td></tr><tr><td>UltraPure 0.5M EDTA, pH 8.0</td><td>ThermoFisher</td><td>15575020</td><td></td></tr><tr><td>PBS, pH 7.4, Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; free</td><td>ThermoFisher</td><td>10010023</td><td></td></tr><tr><td>1.0 M NaOH (1.0N) solution</td><td>SIGMA-ALDRICH</td><td>S2567</td><td>For Melt-off solution. Molecular grade</td></tr><tr><td>Eppendorf LoBind Tubes, 1.5 mL</td><td>Fisher Scientific</td><td>13-698-791</td><td></td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>ELGA PURELAB Flex 3 Water Purification System or&lt;br/&gt; Equivalent 18 M&amp;Omega; water system</td><td>ThermoFisher</td><td>4474524</td><td></td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>PicoPLEX WGA Amplification buffer</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td></tr><tr><td>PicoPLEX WGA Amplification enzyme</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td></tr><tr><td>Ion OneTouch Amplification Plate</td><td></td><td></td><td>In kit: Ion OneTouch 2 Supplies (Part No. A26367). Extended kit component in Sheet 5</td></tr><tr><td>Ion PI Annealing Buffer</td><td></td><td></td><td></td></tr><tr><td>MyOne Beads Capture Solution</td><td></td><td></td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer instrument</td><td>Agilent</td><td>G2939AA</td><td></td></tr><tr><td>Ion OneTouch Breaking Solution (black cap)</td><td></td><td></td><td>In kit: Ion PI Hi?Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5</td></tr><tr><td>Dynabeads MyOne Streptavidin C1</td><td>ThermoFisher</td><td>65001</td><td></td></tr><tr><td>PicoPLEX WGA Cell extraction buffer</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-00.</td></tr><tr><td>PicoPLEX WGA Cell extraction enzyme</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td></tr><tr><td>Ion PI Chip Kit v3</td><td>ThermoFisher</td><td>A26771</td><td></td></tr><tr><td>Ion Chip Minifuge, 230 V</td><td>ThermoFisher</td><td>4479673</td><td></td></tr><tr><td>Ion PI dATP</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion PI dCTP</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion PI dGTP</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>ThermoQ&amp;ndash;Temperature Dry Bath</td><td>TAMAR</td><td>HB-T2-A</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England Biolabs</td><td>M0348L</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England Biolabs</td><td>M0348L</td><td></td></tr><tr><td>Ion PI dTTP</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion OneTouch 2 Instrument</td><td>ThermoFisher</td><td>INS1005527</td><td>ThermoFisher Catalog number: 4474778.</td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>Ion One Touch ES</td><td>ThermoFisher</td><td>8441-22</td><td>ThermoFisher Catalog number: 4469495. Extended kit component in Sheet 5</td></tr><tr><td>Ethanol</td><td>SIGMA-ALDRICH</td><td>51976</td><td>This can be replaced by any brand&#039;s molecular grade absolute ethanol.</td></tr><tr><td>PicoPLEX WGA Extraction enzyme dilution buffer</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-01.</td></tr><tr><td>PicoPLEX WGA Extraction enzyme dilution buffer</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-02.</td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>ThermoFisher</td><td>Q33216</td><td>This model has been replaced by Qubit 4 Fluorometer, Catalog number: Q33226.</td></tr><tr><td>Qubit ds DNA HS Assay kit</td><td>ThermoFisher</td><td>M2002-02</td><td></td></tr><tr><td>Qubit Assay Tubes</td><td>ThermoFisher</td><td>Q32856</td><td></td></tr><tr><td>Ion PI Foaming Solution</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Index for barcoding of libraries</td><td>BaseCare</td><td></td><td>this is a in-house prepared index. Users can buy commercial product from ThermoFisher Ion Xpress Barcode Adapters Kits (Cat. No. 4474517)</td></tr><tr><td>Ion PI Loading Buffer</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Solid(TM) Buffer Kit-1X Low TE Buffer</td><td>ThermoFisher</td><td>4389764</td><td></td></tr><tr><td>Agencour AMPure XP Kit</td><td>Beckman Coulter</td><td>A63880</td><td></td></tr><tr><td>DynaMag-2 magnet (magnetic rack)</td><td>ThermoFisher</td><td>12321D</td><td></td></tr><tr><td>Ion PI Master Mix PCR buffer</td><td></td><td></td><td></td></tr><tr><td>Sorvall Legend Micro 17 Microcentrifuge</td><td>Micro 17</td><td>75002430</td><td></td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>Nuclease-free water</td><td>ThermoFisher</td><td>AM9922</td><td>This can be replaced by other brand.</td></tr><tr><td>PicoPLEX WGA Nuclease-free water</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td></tr><tr><td>Ion OneTouch Oil bottle</td><td></td><td></td><td>Ion PI Hi?Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5</td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td>Extended kit component in Sheet 3</td></tr><tr><td>double-strand DNA standard</td><td></td><td></td><td>This is a in-house prepared DNA standard for calibration of Qubit before quantification of library.</td></tr><tr><td>PicoPLEX WGA Preamplification buffer</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td></tr><tr><td>PicoPLEX WGA Preamplification enzyme</td><td>Rubicon Genomics</td><td>R30050</td><td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td></tr><tr><td>Library Amplification Primer Mix</td><td>ThermoFisher</td><td>4471252</td><td>Extended kit component in Sheet 3</td></tr><tr><td>Ion OneTouch Reaction Filter</td><td></td><td></td><td>Extended kit component in Sheet 5</td></tr><tr><td>Recovery Router</td><td></td><td></td><td>Extended kit component in Sheet 5</td></tr><tr><td>Recovery Tubes</td><td></td><td></td><td>Extended kit component in Sheet 5</td></tr><tr><td>ISP Resuspension Solution</td><td></td><td></td><td></td></tr><tr><td>Ion Proton</td><td>ThermoFisher</td><td>DA8600</td><td>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.</td></tr><tr><td>Ion PI Hi?Q Sequencing Polymerase</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion PI Sequencing Primer</td><td></td><td></td><td></td></tr><tr><td>server for sequencer</td><td>Lenovo</td><td>T260</td><td></td></tr><tr><td>Ion PI Sphere Particles</td><td></td><td></td><td></td></tr><tr><td>Platinum PCR SuperMix High Fidelity</td><td>ThermoFisher</td><td>4471252</td><td></td></tr><tr><td>Nalgene 25mm Syringe Filters</td><td>ThermoFisher</td><td>724-2045</td><td>Pore size: 0.45&amp;mu;m. Specifically for aqueous fluids.</td></tr><tr><td>Ion PI Hi?Q W2 Solution</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion PI 1X W3 Solution</td><td>ThermoFisher</td><td>A26772</td><td></td></tr><tr><td>Ion OneTouch Wash Solution C1</td><td></td><td></td><td></td></tr><tr><td>The Ion PGM Hi?Q View Sequencing Kit</td><td>ThermoFisher</td><td>A30044</td><td>Extended kit component in Sheet 2</td></tr><tr><td>Ion Plus Fragment Library Kit</td><td>ThermoFisher</td><td>4471252</td><td>Extended kit component in Sheet 3</td></tr><tr><td>Ion PI Hi-Q Sequencing 200 Kit (1 sequencing run per initialization)</td><td>ThermoFisher</td><td>A26772</td><td>Extended kit component in Sheet 4</td></tr><tr><td>Ion PI Hi?Q OT2 200 Kit</td><td>ThermoFisher</td><td>A26434</td><td>Extended kit component in Sheet 5</td></tr></tbody></table>

semiconductor sequencing for preimplantation genetic testing for aneuploidy

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

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&#8722;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&#8722;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.

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...

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 ...

semiconductor-sequencing-for-preimplantation-genetic-testing-for

Research

JoVE Journal

Genetics

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis 

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Single Oocyte Bisulfite Mutagenesis

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5–2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

Genotyping of Sea Anemone during Early Development

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

This protocol proposes holistic laboratory procedures required to detect single nucleotide polymorphisms in gastric cancer samples based on an ion semiconductor sequencing platform. The target sequences, ligating adapters, library amplification and purification, and quality control criteria are also described in detail.

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methyl-binding DNA capture Sequencing for Patient Tissues

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Learn semiconductor sequencing for preimplantation genetic testing for aneuploidy using embryo samples. This rapid, cost-effective method delivers reliable genome sequencing with high throughput and results within five days.

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

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