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。
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。
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。
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?…
The authors have nothing to disclose.
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) 。
PCR tubes, 0.2 mL | Axygen | PCR-02D-C | |
UltraPure 0.5M EDTA, pH 8.0 | ThermoFisher | 15575020 | |
PBS, pH 7.4, Ca2+ and Mg2+ free | ThermoFisher | 10010023 | |
1.0 M NaOH (1.0N) solution | SIGMA-ALDRICH | S2567 | For Melt-off solution. Molecular grade |
Eppendorf LoBind Tubes, 1.5 mL | Fisher Scientific | 13-698-791 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
ELGA PURELAB Flex 3 Water Purification System or Equivalent 18 MΩ water system |
ThermoFisher | 4474524 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
PicoPLEX WGA Amplification buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
PicoPLEX WGA Amplification enzyme | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Ion OneTouch Amplification Plate | In 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 instrument | Agilent | G2939AA | |
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 C1 | ThermoFisher | 65001 | |
PicoPLEX WGA Cell extraction buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-00. |
PicoPLEX WGA Cell extraction enzyme | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Ion PI Chip Kit v3 | ThermoFisher | A26771 | |
Ion Chip Minifuge, 230 V | ThermoFisher | 4479673 | |
Ion PI dATP | ThermoFisher | A26772 | |
Ion PI dCTP | ThermoFisher | A26772 | |
Ion PI dGTP | ThermoFisher | A26772 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
ThermoQ–Temperature Dry Bath | TAMAR | HB-T2-A | |
NEBNext dsDNA Fragmentase | New England Biolabs | M0348L | |
NEBNext dsDNA Fragmentase | New England Biolabs | M0348L | |
Ion PI dTTP | ThermoFisher | A26772 | |
Ion OneTouch 2 Instrument | ThermoFisher | INS1005527 | ThermoFisher Catalog number: 4474778. |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
Ion One Touch ES | ThermoFisher | 8441-22 | ThermoFisher Catalog number: 4469495. Extended kit component in Sheet 5 |
Ethanol | SIGMA-ALDRICH | 51976 | This can be replaced by any brand's molecular grade absolute ethanol. |
PicoPLEX WGA Extraction enzyme dilution buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-01. |
PicoPLEX WGA Extraction enzyme dilution buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-02. |
Qubit 3.0 Fluorometer | ThermoFisher | Q33216 | This model has been replaced by Qubit 4 Fluorometer, Catalog number: Q33226. |
Qubit ds DNA HS Assay kit | ThermoFisher | M2002-02 | |
Qubit Assay Tubes | ThermoFisher | Q32856 | |
Ion PI Foaming Solution | ThermoFisher | A26772 | |
Index for barcoding of libraries | BaseCare | this is a in-house prepared index. Users can buy commercial product from ThermoFisher Ion Xpress Barcode Adapters Kits (Cat. No. 4474517) | |
Ion PI Loading Buffer | ThermoFisher | A26772 | |
Solid(TM) Buffer Kit-1X Low TE Buffer | ThermoFisher | 4389764 | |
Agencour AMPure XP Kit | Beckman Coulter | A63880 | |
DynaMag-2 magnet (magnetic rack) | ThermoFisher | 12321D | |
Ion PI Master Mix PCR buffer | |||
Sorvall Legend Micro 17 Microcentrifuge | Micro 17 | 75002430 | |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | |
Nuclease-free water | ThermoFisher | AM9922 | This can be replaced by other brand. |
PicoPLEX WGA Nuclease-free water | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Ion OneTouch Oil bottle | Ion PI Hi‑Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5 | ||
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | Extended kit component in Sheet 3 |
double-strand DNA standard | This is a in-house prepared DNA standard for calibration of Qubit before quantification of library. | ||
PicoPLEX WGA Preamplification buffer | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
PicoPLEX WGA Preamplification enzyme | Rubicon Genomics | R30050 | This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03. |
Library Amplification Primer Mix | ThermoFisher | 4471252 | Extended kit component in Sheet 3 |
Ion OneTouch Reaction Filter | Extended kit component in Sheet 5 | ||
Recovery Router | Extended kit component in Sheet 5 | ||
Recovery Tubes | Extended kit component in Sheet 5 | ||
ISP Resuspension Solution | |||
Ion Proton | ThermoFisher | DA8600 | 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. |
Ion PI Hi‑Q Sequencing Polymerase | ThermoFisher | A26772 | |
Ion PI Sequencing Primer | |||
server for sequencer | Lenovo | T260 | |
Ion PI Sphere Particles | |||
Platinum PCR SuperMix High Fidelity | ThermoFisher | 4471252 | |
Nalgene 25mm Syringe Filters | ThermoFisher | 724-2045 | Pore size: 0.45μm. Specifically for aqueous fluids. |
Ion PI Hi‑Q W2 Solution | ThermoFisher | A26772 | |
Ion PI 1X W3 Solution | ThermoFisher | A26772 | |
Ion OneTouch Wash Solution C1 | |||
The Ion PGM Hi‑Q View Sequencing Kit | ThermoFisher | A30044 | Extended kit component in Sheet 2 |
Ion Plus Fragment Library Kit | ThermoFisher | 4471252 | Extended kit component in Sheet 3 |
Ion PI Hi-Q Sequencing 200 Kit (1 sequencing run per initialization) | ThermoFisher | A26772 | Extended kit component in Sheet 4 |
Ion PI Hi‑Q OT2 200 Kit | ThermoFisher | A26434 | Extended kit component in Sheet 5 |