In order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes. Typically, haplotype resolution is obtained by genotyping multiple polymorphic markers in both parents and a proband or a relative. Sometimes, single sperm typing, or tests on the polar bodies may also be useful. Nevertheless, this process is time-consuming. At present, there was no simple linkage analysis strategy for patients without affected relatives. To solve this problem, we established a haplotyping by linked-read sequencing (HLRS) method without the requirement for additional relatives. First, the haplotype of the genetic disease carriers in the family was constructed by linked-read sequencing, and then the informative single nucleotide polymorphisms (SNPs) in upstream and downstream mutation region were selected to construct the embryo haplotype and to determine whether the embryo was carrying the mutation. Two families were selected to validate this method; one with alpha thalassemia and the other with NDP gene disorder. The haplotyping by linked-read sequencing (HLRS) method was successfully applied to construct parental haplotypes without recruiting additional family members; the method was also validated for PGT-M. The mutation carriers in these families were sequenced by linked-read sequencing, and their haplotypes were successfully phased. Adjacent SNPs of the mutation gene were identified. The informative SNPs were chosen for linkage analyses to identify the carrier embryos. For the alpha thalassemia family, a normal blastocyst was transferred to the uterus and the accuracy of PGT-M was confirmed by amniocentesis at 16 weeks of gestation. Our results suggest that HLRS can be applied for PGT-M of monogenic disorders or de novo mutations where the mutations haplotype cannot be determined due to absence of affected relatives.

中文翻译：

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遗传疾病携带者的链接阅读测序（HLRS）单倍型，无需先证者或亲属即可进行植入前基因测试。

为了减轻等位基因缺失（ADO）的风险并确保单基因疾病（PGT-M）的植入前基因测试的准确性，有必要构建亲本单倍型。通常，通过对父母和先证者或亲戚中的多个多态性标记进行基因分型来获得单倍型分辨率。有时，单精子分型或对极体的检测也可能有用。但是，此过程很耗时。目前，尚无针对未受影响亲属的患者的简单连锁分析策略。为解决此问题，我们建立了通过链接阅读测序（HLRS）方法进行单倍型分型的方法，而无需其他亲戚。首先，通过连锁阅读测序构建家庭遗传疾病携带者的单倍型，然后选择上游和下游突变区域中信息丰富的单核苷酸多态性（SNP）来构建胚胎单倍型并确定胚胎是否携带突变。选择了两个族来验证该方法；一个患有α地中海贫血，另一个患有NDP基因疾病。通过连锁阅读测序（HLRS）方法进行单倍型分析已成功应用于构建父母单倍型，而无需招募其他家庭成员。该方法还针对PGT-M进行了验证。通过连锁阅读测序对这些家族中的突变载体进行测序，并成功地对其单倍型进行分期。确定了突变基因的相邻SNP。选择信息丰富的SNP进行连锁分析，以鉴定携带者的胚胎。对于阿尔法地中海贫血家庭，正常的胚泡被转移到子宫，并且在妊娠16周时通过羊膜穿刺术证实了PGT-M的准确性。我们的结果表明，HLRS可用于单基因疾病或从头突变的PGT-M，其中由于缺乏受影响的亲属而无法确定突变单倍型。