Zhang Zhiqiang, Kang Kai, Xu Linan, Li Xiaolan, He Shujing, Xu Ruixia, Jia Lei, Zhang Shihui, Su Wenlong, Sun Peng, Gu Mengnan, Shan Wenqi, Zhang Yawen, Kong Lingyin, Liang Bo, Fang Cong, Ren Zi
Reproductive Medicine Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; Guangdong Engineering Technology Research Center of Fertility Preservation, Guangzhou, China; Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Basecare Medical Device Co., Ltd., Suzhou, China.
Reprod Biomed Online. 2025 Jan;50(1):104328. doi: 10.1016/j.rbmo.2024.104328. Epub 2024 Jun 21.
Is there a precise and efficient haplotyping method to expand the application of preimplantation genetic testing (PGT)?
In this study, eight cell-line families and 18 clinical families including 99 embryos were used to construct whole-genome haplotyping based on link-read sequencing (Phbol-seq) and optimized analytical workflow with a correction algorithm. The correction algorithm was based on a differentiation of assembly errors and homologous recombination, in which the main feature of parental assembly error was that all embryos (embryo number ≥2) had breakpoints at the same chromosome position.
With Phbol-seq, parental assembly errors and homologous recombination were accurately distinguished and corrected. Using the link-reads (>25% long-reads were ≥30 kilobases [kb]), complete genome-wide parental haplotypes were constructed, and the consistency of the typing results of each chromosome with a conventional method requiring other family members was more than 95%. In addition, the length of N50 contigs was 11.03-16.2 million bases (mb), which was far beyond the N50 contigs from long-read sequencing (148-863 kb). The complete haplotype analysis of all embryos could be performed by Phbol-seq and revealed 100% concordance with the available diagnostic results obtained by the conventional method requiring other family members.
Phbol-seq has high clinical value as a precise and cost-efficient whole-genome haplotyping method without probands as part of PGT and other genetic research, which could promote the application of PGT to decrease the birth of children with genetic diseases and the development of linkage-related genetic research.
是否存在一种精确且高效的单倍型分型方法来扩大植入前基因检测（PGT）的应用？
在本研究中，使用了8个细胞系家族和18个临床家族（包括99个胚胎），基于连接读取测序（Phbol-seq）构建全基因组单倍型，并采用带有校正算法的优化分析流程。校正算法基于组装错误和同源重组的区分，其中亲代组装错误的主要特征是所有胚胎（胚胎数量≥2）在相同染色体位置具有断点。
通过Phbol-seq，能够准确区分并校正亲代组装错误和同源重组。使用连接读取（>25%的长读取≥30千碱基[kb]），构建了完整的全基因组亲代单倍型，并且每条染色体的分型结果与需要其他家庭成员的传统方法的一致性超过95%。此外，N50重叠群的长度为11.03 - 1620万个碱基（mb），远远超过长读取测序的N50重叠群（148 - 863 kb）。通过Phbol-seq可以对所有胚胎进行完整的单倍型分析，并且显示与需要其他家庭成员的传统方法获得的现有诊断结果100%一致。
作为一种精确且具有成本效益的全基因组单倍型分型方法，Phbol-seq在PGT及其他基因研究中无需先证者具有很高的临床价值，这可以促进PGT的应用，以减少患有遗传疾病儿童的出生以及连锁相关基因研究的发展。
