Abstract：
BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) using polar body (PB) biopsy offers a clinical benefit by reducing the number of embryo transfers and miscarriage rates but is currently not cost-efficient. Nanopore sequencing technology opens possibilities by providing cost-efficient and fast sequencing results with uncomplicated sample preparation work flows.
METHODS: In this comparative experimental study, 102 pooled PB samples (99 passing QC) from 20 patients were analyzed for aneuploidy using nanopore sequencing technology and compared with array comparative genomic hybridization (aCGH) results generated as part of the clinical routine. Samples were sequenced on a Nanopore MinION machine. Whole-chromosome copy-numbers were called by custom bioinformatic analysis software. Automatically called results were compared to aCGH results.
RESULTS: Overall, 96/99 samples were consistently detected as euploid or aneuploid in both methods (concordance = 97.0%, sensitivity = 0.957, specificity = 1.0, positive predictive value = 1.0, negative predictive value = 0.906). On the chromosomal level, concordance reached 98.7%. Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, and 97 monosomies in 70 aCGH samples.The whole nanopore work flow is feasible in under 5 h (for one sample) with a maximum time of 16 h (for 12 samples), enabling fresh PB-euploid embryo transfer. A material cost of US$ 165 (EUR 150)/sample possibly enables cost-efficient aneuploidy screening.
CONCLUSIONS: This is the first study systematically comparing nanopore sequencing with standard methods for the detection of PB aneuploidy. High concordance rates confirmed the feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient work flow reveals the clinical utility of this technology, making it clinically attractive for PB PGT-A.
METHODS: In this comparative experimental study, 102 pooled PB samples (99 passing QC) from 20 patients were analyzed for aneuploidy using nanopore sequencing technology and compared with array comparative genomic hybridization (aCGH) results generated as part of the clinical routine. Samples were sequenced on a Nanopore MinION machine. Whole-chromosome copy-numbers were called by custom bioinformatic analysis software. Automatically called results were compared to aCGH results.
RESULTS: Overall, 96/99 samples were consistently detected as euploid or aneuploid in both methods (concordance = 97.0%, sensitivity = 0.957, specificity = 1.0, positive predictive value = 1.0, negative predictive value = 0.906). On the chromosomal level, concordance reached 98.7%. Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, and 97 monosomies in 70 aCGH samples.The whole nanopore work flow is feasible in under 5 h (for one sample) with a maximum time of 16 h (for 12 samples), enabling fresh PB-euploid embryo transfer. A material cost of US$ 165 (EUR 150)/sample possibly enables cost-efficient aneuploidy screening.
CONCLUSIONS: This is the first study systematically comparing nanopore sequencing with standard methods for the detection of PB aneuploidy. High concordance rates confirmed the feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient work flow reveals the clinical utility of this technology, making it clinically attractive for PB PGT-A.
摘要：
背景：使用极体（PB）活检的非整倍体（PGT-A）的植入前遗传测试通过减少胚胎移植的数量和流产率提供了临床益处，但目前并不具有成本效益。纳米孔测序技术通过提供具有成本效益和快速的测序结果以及简单的样品制备工作流程而打开了可能性。
方法：在本比较实验研究中，使用纳米孔测序技术分析来自20名患者的102个汇集的PB样品(99个通过QC)的非整倍性,并与作为临床常规的一部分产生的阵列比较基因组杂交(aCGH)结果进行比较。在NanoporeMinION机器上对样品进行测序。通过定制生物信息学分析软件调用全染色体拷贝数。将自动调用的结果与aCGH结果进行比较。
结果：总体而言，在两种方法中，96/99个样品始终检测为整倍体或非整倍体（一致性=97.0％，敏感性=0.957，特异性=1.0，阳性预测值=1.0，阴性预测值=0.906）。在染色体水平上,一致性达到98.7%。在这项试验中分析的染色体非整倍体覆盖了所有23个染色体和98个三体，和70个aCGH样品中的97个单体。整个纳米孔工作流程在5小时内（对于一个样品）是可行的，最大时间为16小时（对于12个样品），使新鲜的PB-整倍体胚胎移植。165美元(150欧元)/样品的材料成本可能实现成本有效的非整倍性筛选。
结论：这是第一个系统地比较纳米孔测序与检测PB非整倍性的标准方法的研究。高一致率证实了纳米孔技术用于该应用的可行性。此外,快速且具有成本效益的工作流程揭示了该技术的临床实用性，使其对PBPGT-A具有临床吸引力
方法：在本比较实验研究中，使用纳米孔测序技术分析来自20名患者的102个汇集的PB样品(99个通过QC)的非整倍性,并与作为临床常规的一部分产生的阵列比较基因组杂交(aCGH)结果进行比较。在NanoporeMinION机器上对样品进行测序。通过定制生物信息学分析软件调用全染色体拷贝数。将自动调用的结果与aCGH结果进行比较。
结果：总体而言，在两种方法中，96/99个样品始终检测为整倍体或非整倍体（一致性=97.0％，敏感性=0.957，特异性=1.0，阳性预测值=1.0，阴性预测值=0.906）。在染色体水平上,一致性达到98.7%。在这项试验中分析的染色体非整倍体覆盖了所有23个染色体和98个三体，和70个aCGH样品中的97个单体。整个纳米孔工作流程在5小时内（对于一个样品）是可行的，最大时间为16小时（对于12个样品），使新鲜的PB-整倍体胚胎移植。165美元(150欧元)/样品的材料成本可能实现成本有效的非整倍性筛选。
结论：这是第一个系统地比较纳米孔测序与检测PB非整倍性的标准方法的研究。高一致率证实了纳米孔技术用于该应用的可行性。此外,快速且具有成本效益的工作流程揭示了该技术的临床实用性，使其对PBPGT-A具有临床吸引力
