Name: semiconductor sequencing for preimplantation genetic testing for aneuploidy
Uploaded: 2019-08-25T13:00:00
Description: Watch this Scientific Journal Video about Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy at JoVE.com

这项研究得到了香港综合研究基金（参考文献号14162417）、国家自然科学基金（参考文献号81860272）、广西省科学技术基金重大研究计划（参考文献号81860272）的支持。AB16380219）和中国博士后科学基金资助（参考号2018M630993） 。

香港中文大学-新界东集群临床研究伦理委员会（参考编号：2010.432）获道德认可。研究许可证获香港人类生殖科技委员会批准（编号R3004）。
1. 全基因组扩增
<ol><li>开始之前，检查磁珠（材料表）的体积，以确保每个样品的磁珠量不低于135μL（超过20%）。将磁珠保持在室温（RT）至少30分钟。为每个样品制备720μL（多20%）乙醇。在105°C下为热循环器（材料?...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+microarray-based+24+chromosome+aneuploidy+screening+demonstrates+that+cleavage-stage+FISH+poorly+predicts+aneuploidy+in+embryos+that+develop+to+morphologically+normal+blastocysts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+aneuploid+embryos+undergo+self-correction+in+correlation+with+their+developmental+potential.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+cytogenetic+analysis+of+the+human+blastocyst+stage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+next-generation+sequencing-based+protocol+for+24-chromosome+aneuploidy+screening+of+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+next-generation+sequencing+technology+for+comprehensive+aneuploidy+screening+of+blastocysts+in+clinical+preimplantation+genetic+screening+cycles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utilisation+of+a+rapid+low-pass+whole+genome+sequencing+technique+for+the+diagnosis+of+aneuploidy+in+human+embryos+prior+to+implantation.">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. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESHRE+PGD+Consortium/Embryology+Special+Interest+Groupbest+practice+guidelines+for+polar+body+and+embryo+biopsy+for+preimplantation+genetic+diagnosis/screening+(PGD/PGS).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comparison+of+the+performance+of+four+whole+genome+amplification+kits+on+ion+proton+platform+in+copy+number+variation+detection.">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>Munn&#233;, S., Grifo, J., Wells, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaicism:+&#8220;survival+of+the+fittest&#8221;+versus+&#8220;no+embryo+left+behind&#8221;.">Mosaicism: &#8220;survival of the fittest&#8221; versus &#8220;no embryo left behind&#8221;.</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+of+chromosome+abnormality+affects+embryo+morphology+dynamics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESHRE+PGD+consortium+best+practice+guidelines+for+amplification-based+PGD.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shallow+whole+genome+sequencing+is+well+suited+for+the+detection+of+chromosomal+aberrations+in+human+blastocysts.">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. 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与其他测序化学不同，此处描述的定序器使用半导体检测核苷酸。芯片本身是一种电子设备，通过聚合酶驱动的碱基结合17检测氢离子，使质子计划的测序时间达到2⁄4小时。此外，该芯片是一种微孔芯片，允许定位一个目标分子，这不同于其他测序器的流细胞测序化学。该协议是针对 PGT-A 应用优化的经过修改的协议。优化包括通过酶法对扩增DNA进行碎片化，而不是声波，以减?...

选择具有正常染色体拷贝数（双发体）的优质活胚胎进行辅助生殖转移，有助于改善妊娠结局。传统上，成熟的形态分级系统由于其易于获得和非侵入性而被广泛用于胚胎评估。然而，已经表明，形态评估只能提供关于胚胎质量1和植入潜力2的有限信息。一个根本原因是它不能评估胚胎的染色体组成。
染色体非倍体（染色体的异常拷贝数）是导致胚胎发育停止、植入失败或妊娠损失的主要原因之一。非倍体的发生在人类胚胎中已有所记载，在裂解阶段胚胎中占60%-70%，在胚泡5中分别占60%-70%和50%~60%。这在一定程度上造成了提高体外受精（IVF）治疗妊娠率的瓶颈，目前这一比率一直维持在35%-40%左右。因此，选择幼倍体胚胎进行移植被认为有利于改善妊娠结局。为此，进一步开发了非倍体（PGT-A）的植入前基因测试，以利用基因方法研究胚胎的生存能力。支持PGT-A关键作用的随机对照试验和队列研究越来越多。实践证明，PGT-A的应用降低了流产率，提高了临床妊娠率和植入率8，持续妊娠率和活产率9.
从历史上看，PGT-A应用了不同的方法，如荧光原位杂交（FISH）、比较基因组杂交（CGH）、阵列-CGH和单核苷酸多态性（SNP）微阵列。先前的研究表明，FISH用于裂解阶段胚胎的PGT-A结果与使用59273array-CGH或SNP 微阵列5927310.这些差异可归因于染色体马赛克，FISH技术伪影，或胚胎自我纠正的染色体分离错误在开发11。人们普遍认为，使用胚泡营养素（TE）活检阵列为基础的PGT-A，如阵列-CGH或SNP-微阵列，对识别胚胎10、12的染色体不平衡有效。最近，单细胞下一代测序（NGS）为基因分析提供了高通量和具有成本效益的方法，并在PGT-A 13、14、15中显示出临床应用能力，这使得它成为有望替代现有方法。
在这里，我们提出了一种快速、可靠和低成本的基于半导体测序的NGS方法，用于筛选人类胚胎中的非倍体。工作流程的第一步是活检胚胎标本的全基因组扩增（WGA），使用单细胞WGA试剂盒，然后构建测序库，随后在半导体测序系统上进行测序。
通过检测DNA链合成过程中从每个脱氧核苷三磷酸结合中释放的H+离子，系统传输半导体元件捕获的化学信号（pH变化），以直接提供数字数据，进一步解释为DNA序列信息。这种简单的测序化学方法消除了昂贵的光学检测和复杂测序反应的要求，降低了总试剂成本，并将测序运行时间缩短为2⁄4小时16小时。更重要的是，根据制造商的性能规格，半导体测序平台每次运行可以生成多达 15 GB 的测序数据（取决于库的质量），这明显高于其他一些测序器仅生成约 3⁄4 GB 数据（读取长度为 2 x 75 bp）17。在PGT-A的临床应用中，该平台可实现每个芯片24个样本，产生多达8000万次读取17次，每个样品至少100万次唯一读取。读取深度可以确保每个样本至少具有0.05倍的全基因组覆盖率。该平台的上述优点使其成为理想的筛选方法，从而方便了其在PGT-A18中的广泛应用。

<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, Ca2+ and Mg2+ 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 
			Equivalent 18 M&#937; 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&#8209;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&#8211;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&#39;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><a href="https://www.thermofisher.com/order/catalog/product/AM9922">AM9922</a></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&#8209;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&#8209;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&#956;m. Specifically for aqueous fluids.</td>
		</tr>
		<tr>
			<td>Ion PI Hi&#8209;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&#8209;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&#8209;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

染色体非倍体是导致胚胎发育停止、植入失败或妊娠丧失的主要原因之一，在人类胚胎中已有详细记载。非倍体（PGT-A）的植入前基因测试是一种基因测试，通过检测胚胎的染色体异常，显著改善生殖结果。下一代测序（NGS）为基因分析提供了高通量和具有成本效益的方法，并在PGT-A中显示出临床应用性。在这里，我们提出了一种快速和低成本的基于半导体测序的NGS方法，用于筛选胚胎中的非倍体。工作流程的第一步是活检胚胎标本的全基因组扩增（WGA），然后是测序库的构建，以及随后对半导体测序系统的测序。通常，对于 PGT-A 应用程序，可以在每个芯片上加载和排序 24 个样本，以平均读取长度为 150 个碱基对生成 60-8000 万次读取。该方法为执行序列库的模板放大和扩充提供了精细的协议，使PGT-A检测可重复、高通量、经济高效和省时。该半导体音序器的运行时间仅为2~4小时，将接收样品至发布报告的周转时间缩短至5天。所有这些优点使得这种测定成为从胚胎检测染色体非倍体的理想方法，从而有助于它在PGT-A中的广泛应用。

基于这一修改协议，首次将半导体测序平台应用于PGT-A。我们测试了裂解阶段胚泡和胚泡阶段胚胎的活检。建议活检细胞尽快进行WGA，以防止DNA降解。以前的研究比较了不同WGA方法的性能，指出我们在这里描述的方法在100KB20的bin大小下具有最佳的均匀性。考虑到均匀性和中位数绝对成对差（MAPD）21的性能，使用半导体定序器为PGT-A选择?...

Watch this Scientific Journal Video about Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy at JoVE.com

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

这里介绍了一种半导体测序方法，用于非倍体（PGT-A）的植入前基因检测，具有周转时间短、成本低、通量高等优点。

用于抗倍体植入前基因测试的半导体测序

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

我们在这里介绍的协议是一种快速和低成本的半导体，基于测序的方法，用于筛选胚胎中的非倍体。执行模板扩增的精炼协议以及测序库的排列使PGTA检测具有可重复性、高吞吐量、成本效益和节省时间。此半导体音序器的运行时间只有两到四个小时。将从接收样品缩短为五天的周转时间。此方法为非倍体筛选、拷贝数变异和单核苷酸多态性调用提供基因组测序。由于不同的测序化学，此协议编写的库不能直接在其他测序系统上排序。但同一的测序器，但不同的库准备，它能够做非侵入性产前筛查。演示该程序将是郭湘春，一个技术员从我们实验室。要开始测序，每个稀释库的涡流，并在微型离心机上短暂旋转四次，每次旋转三秒钟。然后，采取每个库的五微升，池入无核，一点五毫升管。漩涡混合库，在迷你离心机上短暂旋转三秒钟。将 150 微升的破碎溶液添加到两个新的恢复管中。安装新的恢复管、恢复路由器和放大板。将油瓶倒置三次。混合。确保机油和回收液至少已满三分之二。旋转主混合PCR缓冲液30秒，在迷你离心机上短暂旋转3秒钟。旋转球体颗粒和混合库一分钟，并在微型离心机上短暂旋转三秒钟。接下来，准备2400微升结扎混合，通过添加172微升无核酸酶，8微升的混合库，120微升的酶混合物，和100微升的球体颗粒，到管内含有2000微升的主混合PCR缓冲液。将移液器设置为 800 微升，并通过样品端口将连接混合物加载到反应过滤器中。将油瓶倒置三次，使用 P1000 移液器将 200 微升反应油添加到反应过滤器中。选择程序质子，然后选择辅助按钮，以确保设备已正确设置，按照显示器上的说明。然后单击"下一步"以启动程序。将乳液PCR产品样品装载100微升，洗涤珠130微升，ES洗涤溶液300微升，将熔离溶液装入8管条。将八根管条放在浓缩系统中。安装移液器尖端和新的零点两毫升管，然后启动程序。将零点两毫升管在 15，500 倍 G 下离心五分钟。丢弃上一杯，并保留十微升的浓缩产品。将200微升无核酸水加入管中，通过涡流混合。再次将零点两毫升管离心。丢弃上一杯，并保留10微升的浓缩产品。将 90 微升无核酸水加入管中，通过涡流混合。要准备模板，请旋转正控并短暂旋转。在浓缩产品的100微升中加入5个正控微升。涡流和离心机在15，500次G下5分钟。丢弃上一点，并保留模板的 10 微升。接下来，向模板管添加 20 微升测序底片和 15 微升退火缓冲液。旋转管，在迷你离心机上短暂旋转三秒钟。用加热盖将管子孵育在热循环器中。然后向管中加入10微升的装载缓冲液。通过上下移液混合。通过上下移液将 55 微升样品混合，将样品加载到芯片的加载井中。保持用过的移液器尖端和零点两毫升PCR管。当一些样品进入芯片时，将芯片放在芯片微型离心机上。检查位置并离心芯片 10 分钟。通过将500微升退火缓冲液和500微升无核酸酶水加入一点5毫升管中，准备50%退火缓冲液。准备冲洗溶液，将500微升退火缓冲液和500微升100%2-丙醇加入一点5毫升管中。然后，准备发泡混合物，将 50% 退火缓冲液的 49 微升和发泡溶液的一微升添加到两个新的一点五毫升管中。移液器空气进入其中一个发泡混合物，并加载120微升的气泡到装载井。将过量排出的液体从出口井转移到装载井，并在芯片微型离心机上离心30秒。对第二个发泡混合物重复该工艺。将 50% 退火缓冲液的 55 微升添加到零点两毫升管中。使用保持的移液器尖端上下移液器。将退火缓冲液的所有 55 微升加载到装载井。在指定的芯片微型离心机上将芯片离心 30 秒。将冲洗溶液的100微升装入芯片装载井，然后从出口井中丢弃排出的液体。重复此加载步骤一次。将100微升的50%退火缓冲液装进芯片负载井中。重复此加载步骤共三次。将 50% 退火缓冲液中的 60 微升和测序酶的 6 微升加入新的一点五毫升管中。通过上下移液混合。慢慢移液器65微升这种混合溶液进入芯片加载井，避免气泡。使芯片远离光线，并在室温下孵育五分钟。最后，在孵育后，立即将芯片加载到音序器上，然后单击屏幕上的"开始测序运行"以开始排序。对186个裂解阶段和1135个胚胎进行回顾性统计分析，导致两种样本的WGA成功率超过95%。裂解阶段组的测序质量控制失败率为3.4%，在囊肿阶段组中只有1.9%。除了异常，测序数据还可用于其他分析。例如，大小小于 5 兆基的拷贝数变化，或 mycondrial DNA 分析，具体取决于测序深度和质量。随着拷贝数调用的改进，研究人员可以进一步研究人类早期胚胎中染色体马赛克的未来。

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

胃肠道有机体遗传工程的通用高效电穿孔协议

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the ...