Name: pre-implantation genetic testing for aneuploidy on a semiconductor based next-generation sequencing platform
Uploaded: 2022-08-17T12:50:00
Description: Watch this Scientific Journal Video about Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform at JoVE.com

我们要感谢张勇明博士和侯荣基先生对LIMS扩展应用的建议。本研究由解放军计划生育专项研究项目（17JS008、20JSZ08）、广西代谢性疾病研究重点实验室基金（No.20-065-76）和广州市民健康科技攻关项目（201803010034）资助。

本研究应用的所有方案和滋养外胚层（TE）活检（1.1.1.1部分）均于2017年9月18日获得第924医院人类研究伦理委员会的审查和批准（编号：PLA924-2017-59）。患者/受试者提供了参加本研究的书面知情同意书。
1. 从人胚胎活检和全基因组扩增中分离DNA
<ol><li>全基因组扩增实验方案9，10 注意：微微PLEX WGA试剂盒用于进行全基?...

<ol>
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A., Barad, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+screening+(PGS)+still+in+search+of+a+clinical+application:+a+systematic+review.">Preimplantation genetic screening (PGS) still in search of a clinical application: a systematic review.</a> Reproductive Biology and Endocrinology. 12, 22 (2014).</li><li>Bono, 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=Validation+of+a+semiconductor+next-generation+sequencing-based+protocol+for+pre-implantation+genetic+diagnosis+of+reciprocal+translocations.">Validation of a semiconductor next-generation sequencing-based protocol for pre-implantation genetic diagnosis of reciprocal translocations.</a> Prenatal Diagnosis. 35 (10), 938-944 (2015).</li><li>Handyside, A. 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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=A+quantitative+comparison+of+single-cell+whole+genome+amplification+methods.">A quantitative comparison of single-cell whole genome amplification methods.</a> PLoS One. 9 (8), 105585 (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+pre-implantation+genetic+screening+cycles.">Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical pre-implantation genetic screening cycles.</a> Human Reproduction. 29 (12), 2802-2813 (2014).</li><li>Damerla, R. R., 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=Ion+Torrent+sequencing+for+conducting+genome-wide+scans+for+mutation+mapping+analysis.">Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis.</a> Mammalian Genome. 25 (3-4), 120-128 (2014).</li><li>Brezina, P. R., Anchan, R., Kearns, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+testing+for+aneuploidy:+what+technology+should+you+use+and+what+are+the+differences.">Preimplantation genetic testing for aneuploidy: what technology should you use and what are the differences.</a> Journal of Assisted Reproduction and Genetics. 33 (7), 823-832 (2016).</li><li>Landrum, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mendelian+inheritance+in+man+and+its+online+version,+OMIM.">Mendelian inheritance in man and its online version, OMIM.</a> American Journal of Human Genetics. 80 (4), 588-604 (2007).</li><li>Wang, K., Li, M., Hakonarson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANNOVAR:+functional+annotation+of+genetic+variants+from+high-throughput+sequencing+data.">ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.</a> Nucleic Acids Research. 38 (16), 164 (2010).</li><li>Zhao, M., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNVannotator:+A+comprehensive+annotation+server+for+copy+number+variation+in+the+human+genome.">CNVannotator: A comprehensive annotation server for copy number variation in the human genome.</a> PLoS One. 8 (11), 80170 (2013).</li><li>Zhang, W., 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+application+of+next-generation+sequencing+in+pre-implantation+genetic+diagnosis+cycles+for+Robertsonian+and+reciprocal+translocations.">Clinical application of next-generation sequencing in pre-implantation genetic diagnosis cycles for Robertsonian and reciprocal translocations.</a> Journal of Assisted Reproduction and Genetics. 33 (7), 899-906 (2016).</li><li>Xu, J., 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=Mapping+allele+with+resolved+carrier+status+of+Robertsonian+and+reciprocal+translocation+in+human+pre-implantation+embryos.">Mapping allele with resolved carrier status of Robertsonian and reciprocal translocation in human pre-implantation embryos.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (41), 8695-8702 (2017).</li></ol>

胚胎的染色体非整倍体是大部分妊娠丢失的原因，无论是自然受孕还是 体外 受精（IVF）。在IVF的临床实践中，提出筛选胚胎非整倍体并转移真倍体胚胎可以改善IVF的结果。荧光 原位 杂交是最早用于性别选择和PGT-A的技术;然而，这种技术需要实验室人员更多的技术专长，并且相对劳动密集型。越来越多的使用荧光 原位 杂交的PGT-A研究显示活产率没有改善17<su...

非整倍性是指由于存在一条或多条额外染色体或缺少一条或多条染色体而导致的染色体数量异常。携带某种类型非整倍体的胚胎，例如失去一条X染色体（特纳综合征），额外的常染色体拷贝，如常染色体21（唐氏综合症），13（帕陶综合征）和18（爱德华兹综合征）的三体性，或额外的性染色体，如47，XXY（克林费尔特综合征）和47，XXX（三重X综合征），可以存活到出生缺陷1的足月。非整倍体是妊娠早期流产和 体外 受精（IVF）失败的主要原因2。据报道，在IVF实践中，通过自然周期和药物对照组的不同年龄层，非整倍率可能在25.4%-84.5%之间。
下一代测序技术在临床上基因信息的测定中得到了广泛的应用。它提供了对基因组序列的实用访问，具有高效率和高通量。特别是，下一代测序也彻底改变了遗传因素的疾病诊断和基因组异常性测试4。基于半导体的测序系统使用半导体测序技术将生物反应测序中的化学信号直接传输到数字数据中，从而在3-7小时5，6内提供直接的实时检测数据序列。
在IVF程序中，植入前基因检测（PGT）在移植到子宫之前调查胚胎的遗传特征，以改善IVF结果并降低新生儿遗传疾病的风险1，7。在PGT与NGS技术相结合的情况下，从少于10个细胞中提取的遗传物质被全基因组扩增试剂或独立开发的全基因组扩增试剂扩增。这只需要扩增阶段的一个步骤，不需要预扩增，即可获得全基因组扩增产物。在构建的文库中设计并应用用于拷贝数变异和特殊基因位点测序的引物或面板。
NGS中植入前基因检测非整倍体（PGT-A）的典型工作流程涉及连续程序，并且需要实验室人员的繁重工作量8。一些误操作导致程序回滚可能会导致实验室的时间和资源意外损失。对于PGS-NGS工作流程，简洁明了的标准操作程序（SOP）是有帮助的;但是，字格式协议无法提供有关样品处理、设备操作和仪器设置的更详细信息，这些信息可以在视频协议中可视化。在本文中，经过验证的工作流程与操作细节的可视化演示相结合，可以在半导体测序平台上的PGT实践中提供更直接，更直观的参考协议。
此处的协议描述了一种支持并行批处理多达16个胚胎活检的方法。对于较大的批次，建议使用基于商业试剂盒的半导体测序方案，例如Reproes-PGS。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m Syringe Filter Unit</td>
			<td>Merkmillipore</td>
			<td>Millex-HV</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL DNA LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>30108051</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Greiner Bio-One</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mLDNA LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>30108078</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Anstart Taq Buffer (Mg2+ Plus)</td>
			<td>FAPON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Anstart Tap DNA Polymerase</td>
			<td>FAPON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP reagent (magnetic beads for dna binding)</td>
			<td>Beckman</td>
			<td>A63881</td>
			<td>https://www.beckman.com/reagents/genomic/cleanup-and-size-selection/pcr/a63881</td>
		</tr>
		<tr>
			<td>Cell Lysis buffer</td>
			<td>Southern Medical University</td>
			<td></td>
			<td>Cell lysis buffer containing 40 mM Tris (pH 8), 100 mM NaCl, 2 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% (v/v) Triton X-100, 5 mM sodium pyrophosphate, 2 mM &#946;-glycerophosphate, 0.1% SDS</td>
		</tr>
		<tr>
			<td>ClinVar</td>
			<td>NCBI</td>
			<td></td>
			<td>https://www.ncbi.nlm.nih.gov/clinvar/</td>
		</tr>
		<tr>
			<td>DNA elution buffer</td>
			<td>NEB</td>
			<td>T1016L</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>Vazyme</td>
			<td>P031-AA</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Life Technologies</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Guangzhou Chemical Reagent Factory Thermo Fisher Scientific</td>
			<td></td>
			<td>http://www.chemicalreagent.com/</td>
		</tr>
		<tr>
			<td>Independently developed whole genome amplification reagents</td>
			<td>Southern Medical University</td>
			<td></td>
			<td>The reagents consist of the following components: 
			1. Cell Lysis 
			2. Amplification Pre-mixed solution 
			&#160;&#160;&#160; 1) Primer WGA-P2 (10 &#956;M) 
			&#160;&#160;&#160; 2) dNTP (10 mM) 
			&#160;&#160;&#160; 3) 5x Anstart Taq Buffer (Mg2+ Plus) 
			3. Amplification Enzyme 
			&#160;&#160;&#160; 1) Anstart Tap DNA Polymerase (5 U/&#956;L)</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q OT2 200 Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A26434</td>
			<td>Kit mentioned in step 4.2.8</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q Sequencing 200 Kit&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A26433</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Proton System</td>
			<td>Life Technologies</td>
			<td>4476610</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Reporter Server System</td>
			<td>Life Technologies</td>
			<td>4487118</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>Guangzhou Chemical Reagent Factory</td>
			<td></td>
			<td>http://www.chemicalreagent.com/</td>
		</tr>
		<tr>
			<td>Library Preparation Kit</td>
			<td>Daan Gene Co., Ltd</td>
			<td>114</td>
			<td>https://www.daangene.com/pt/certificate.html</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Life Technologies</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo WGA-P2</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td>5&#39;-ATGGTAGTCCGACTCGAGNNNN 
			NNNNATGTGG-3&#39;</td>
		</tr>
		<tr>
			<td>OneTouch 2 System</td>
			<td>Life Technologies</td>
			<td>4474779</td>
			<td>&#160;Template amplification and enrichment system</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02D-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Kit</td>
			<td>Takara Bio USA</td>
			<td>R300671</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Quality Scientific Products</td>
			<td></td>
			<td>https://www.qsptips.com/products/standard_pipette_tips.aspx</td>
		</tr>
		<tr>
			<td>Portable Mini Centrifuge LX-300</td>
			<td>Qilinbeier</td>
			<td>E0122</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q33216</td>
			<td>Fluorometer</td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer server system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Torrent Suite Software</td>
		</tr>
		<tr>
			<td>Sequencing Reactions Universal Kit</td>
			<td>Daan Gene Co., Ltd</td>
			<td>113</td>
			<td>https://www.daangene.com/pt/certificate.html 
			This kit contains the following components: 
			1. Template Preparation Kit Set 
			 
			1.1 Template Preparation Kit: 
			Emulsion PCR buffer 
			Emulsion PCR enzyme mix 
			Template carrier solution 
			 
			1.2 Template Preparation solutions: 
			Template preparation reaction oil I 
			emulsifier breaking solution II 
			Template Preparation Reaction Oil II 
			Nuclease-free water 
			Tween solution 
			Demulsification solution I 
			Template washing solution 
			C1 bead washing solution 
			C1 bead resuspension solution 
			Template resuspension solution 
			 
			1.3 Template Preparation Materials: 
			Reagent tube I 
			connector 
			Collection tube 
			Reagent tube pipette I 
			Amplification plate 
			8 wells strip 
			Dedicated tips 
			Template preparation washing adapter 
			Template preparation filter 
			 
			2. Sequencing Kit Set 
			 
			2.1 Sequencing Kit: 
			dGTP 
			dCTP 
			dATP 
			dTTP 
			Sequencing enzyme solution 
			Sequencing primers 
			Quality control templates 
			 
			2.2&#160; Sequencing Solutions: 
			Sequencing solution II 
			Sequencing solution IIII 
			Annealing buffer 
			Loading buffer 
			Foaming agent 
			Chlorine tablets 
			C1 bead 
			 
			2.3 Sequencing Materials: 
			Reagent Tube II 
			Reagent tube cap 
			Reagent tube sipper&#160; II 
			Reagent bottle sipper 
			Reagent bottles 
			 
			3. Chip</td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>Sigma</td>
			<td>72068-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Life Technologies</td>
			<td>4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-implantation genetic testing for aneuploidy on a semiconductor based next-generation sequencing platform

下一代测序在确定遗传变异的临床应用中变得越来越重要。在植入前基因检测中，该技术在可扩展性、通量和成本方面具有独特的优势。对于用于非整倍性分析的植入前基因测试，这里介绍的基于半导体的下一代测序（NGS）系统提供了一种全面的方法，以至少8 Mb的分辨率确定结构遗传变异。从样品采集到最终报告，工作流程需要多个步骤，并严格遵守协议。由于各种关键步骤可以决定扩增的结果、文库的质量、读取的覆盖率和数据输出，因此，除文字外，具有视觉演示的描述性信息可以为操作和操作提供更多细节，这可能会对所有关键步骤的结果产生重大影响。本文介绍的方法将显示活检滋养外胚层（TE）细胞的全基因组扩增（WGA），基因组文库构建，测序仪管理以及最终生成拷贝数变异报告所涉及的程序。

当序列计划在机器中运行过程完成后完成时，序列服务器系统会报告摘要，其中包含生成的数据、芯片状态、ISP 加载速率和磁带库质量的描述性信息，如图 2 所示。在结果演示中，获得了总基数中的17.6 G数据，ISP在芯片总井中的总体加载速率为88%;热图显示样品均匀地加载到芯片的总面积上（图2A）。在代表性运行中，总共获得了99，761，079次读取，其?...

Watch this Scientific Journal Video about Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform at JoVE.com

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

该协议介绍了在基于半导体的下一代测序平台上对非整倍性进行植入前基因检测所需的整体实验室内程序。在这里，我们以具有代表性的结果介绍了全基因组扩增、DNA片段选择、文库构建、模板制备和测序工作流程的详细步骤。

基于半导体的下一代测序平台上非整倍体的植入前基因检测

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...

该协议介绍了从单细胞扩增到最终报告数据的过程，用于基于半导体的下一代测序平台上的非整倍性植入前基因检测。该程序可以帮助观察者很好地了解PGT-A测序的实验工作流程，并在具有基于半导体的NGS平台的诊断实验室中复制该技术。首先移取25微升全基因组扩增产物，并将其转移到预装有25微升无核酸酶水的1.5毫升离心管中。涡旋并混合DNA纯化珠。将50微升该溶液等分到每个样品管中。涡旋并短暂离心五秒钟。将试管在室温下放置五分钟以进行DNA结合过程。将1.5毫升离心管插入磁性架。然后，等到所有磁珠都被吸引到管的侧壁上。小心地将上清液转移到新的1.5毫升离心管中。避免移出磁珠。按原样品体积。移液30微升磁珠，涡旋并短暂离心五秒钟。将试管在室温下放置五分钟以进行DNA结合过程。将 1.5 毫米离心管插入磁性架。然后，等到所有磁珠都被吸引到管的侧壁上。小心地除去并丢弃上清液。避免将磁珠移出。将300微升70%乙醇移液到1.5毫升离心管中。然后以 180 度角轻轻旋转试管两次，并沿管壁移动珠子进行彻底清洗。移液并弃去上清液。避免将磁珠移出。重复洗涤程序一次。清洁程序完成后，在系统中放置一个新的扩增板。将收集管（每个收集管预装有 150 微升破碎溶液二）插入转子，然后将桥放在收集管上。将反应油加入管的一半，将乳化剂破碎溶液加入管的三分之一。将用于模板制备的新过滤器放在试管架上，样品入口朝上。涡旋溶液五秒钟，短暂离心五秒钟。然后在移液800微升三次后将溶液转移到过滤器的样品通道中。在最后一次注射之前离心管以减少气泡。避免将空气注入过滤器。注入200微升反应油二，按照混合溶液。向下转动过滤器的样品入口，并用过滤器更换洗涤适应装置。将样品针插入转子盖的中心孔中，然后将针头压到底部。单击屏幕上的“运行”按钮，然后根据相应的套件选择程序。然后选择“辅助”以检查所有步骤。单击“下一步”，直到运行开始。运行大约需要 4.5 小时才能完成。程序完成后选择“下一步”。系统将开始10分钟的离心过程。离心后，单击“打开盖子”按钮并将收集管移动到架子上。如果该过程在 15 分钟内没有进入下一步，请按最终旋转以重新离心。除去收集管中的上清液，在管中留下100微升溶液。通过移液混合剩余的样品，并将样品转移到标有OT的新1.5毫升离心管中。移取上清液时，避免沿侧面接触管底部。将100微升无核酸酶的水移液到每个收集管中，并将溶液转移到通过重复移液洗涤的OT管中。向OT管中加入600微升无核酸酶的水，以构成一毫升的总体积。在离心机中以15， 500倍G涡旋30秒，持续8分钟。轻轻除去上清液，在OT管中留下100微升溶液。使用吸头完全丢弃油层。加入900微升无核酸酶的水，涡旋30秒，以15， 500倍G离心8分钟。除去上清液直到保留20微升溶液，然后加入模板重悬溶液以弥补100微升的体积。然后涡旋30秒，短暂离心两秒钟。在样品处理后 12 小时内进行下一步 将 100 微升稀释库、130 微升 C1 珠、300 微升三 x 模板洗涤溶液和 300 微升熔融脱落溶液加载到八个孔条中。将八个孔条安装在富集模块上，装入移液吸头，并将 200 微升离心管置于收集位置。单击“开始”以运行该程序。吸取55微升样品溶液并将其注入芯片的样品孔中。将芯片放在离心机上。将凹口朝向外部，将样品门户保持在内部。然后，将其与另一个用过的芯片平衡并离心 10 分钟。按照文本手稿中的说明准备加载溶液。将100微升空气吹入发泡溶液中。反复移液，直到气泡处于致密的起泡状态，并将泡沫体积保持在约250微升。离心机后将芯片放在工作台上。将 100 微升泡沫注入样品孔，然后将挤出的溶液取出到出入口中。然后，将芯片短暂离心 30 秒。重复此过程一次。在50%洗涤缓冲液中注入100微升两次，并在每次注射后在出门口中除去挤出的溶液。然后在50%退火缓冲液中注入100微升三次，并在每次注射后在出门中除去挤出的溶液。在50%酶反应缓冲液中注入65微升，避免气泡，并在出门中除去挤出的溶液。将加载的芯片在室温下稳定五分钟，然后将芯片安装在定序器芯片门户上。选择“计划”程序，检查信息并开始运行。代表性运行共获得17.6 GB的数据，离子球粒子在芯片总孔中的总加载率为88%。热图确认了总芯片面积上的均匀样品加载。总读取量的 77% 可用。所有装有ISP的孔均显示出100%的模板富集，其中78%是克隆的。在所有克隆模板中，97%有资格作为最终文库。读取的平均长度为177个碱基对，而中位数和模式分别为176和174个碱基对。模板中胸腺嘧啶、胞嘧啶和腺嘌呤的峰数在合格临界线50上约为76。在所有具有活ISP的可寻址孔中，99.5%符合构建文库的资格，过滤掉20%的多克隆和低质量文库，而76.6%的ISP符合进一步分析的条件。单个样本的拷贝数方差结果描绘了 182.16 兆碱基对马赛克三体，即 P 16.3 至 Q 35.2 在 4 号染色体上，33 点 13 兆碱基对单体片段为 Q 11.1 至 Q 13.33 在 22 号染色体上。进行片段选择时，应根据同一批次样品的大小修改步骤之间的时间间隔，以防止前端和后端样品过度暴露和暴露不足。

Research

JoVE Journal

Genetics

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

科研

遗传学

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

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 ...

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 ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

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 ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

RNA 下一代测序和生物信息学管道, 以识别点对位的表达 Line-1

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...