Zhao Yan, Tsuiko Olga, Jatsenko Tatjana, Peeters Greet, Souche Erika, Geysens Mathilde, Dimitriadou Eftychia, Vanhie Arne, Peeraer Karen, Debrock Sophie, Van Esch Hilde, Vermeesch Joris Robert
Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium.
Centre for Human Genetics, University Hospitals Leuven, Leuven 3000, Belgium.
Nucleic Acids Res. 2025 Mar 20;53(6). doi: 10.1093/nar/gkaf247.
Long-read whole-genome sequencing (lrWGS) enhances haplotyping by providing more phasing information per read compared to short-read sequencing. However, its use for single-cell haplotype phasing remains underexplored. This proof-of-concept study examines lrWGS data from single cells for small variant (single nucleotide variant (SNV) and indel) and structural variation (SV) calling, as well as haplotyping, using the Genome in a Bottle (GIAB) Ashkenazi trio. lrWGS was performed on single-cell (1 cell) and multi-cell (10 cells) samples from the offspring. Chromosome-length haplotypes were obtained by leveraging both long reads and pedigree information. These haplotypes were further refined by replacing them with matched parental haplotypes. In single-cell and multi-cell samples, 92% and 98% of heterozygous SNVs, and 74% and 78% of heterozygous indels were accurately haplotyped. Applied to human embryos for preimplantation genetic testing (PGT), lrWGS demonstrated 100% consistency with array-based methods for detecting monogenic disorders, without requiring phasing references. Aneuploidies were accurately detected, with insights into the mechanistic origins of chromosomal abnormalities inferred from the parental unique allele fractions (UAFs). We show that lrWGS-based concurrent haplotyping and aneuploidy profiling of single cells provides an alternative to current PGT methods, with applications potential in areas such as cell-based prenatal diagnosis and animal and plant breeding.
与短读长测序相比，长读长全基因组测序（lrWGS）通过每条读长提供更多的定相信息来增强单倍型分型。然而，其在单细胞单倍型定相中的应用仍未得到充分探索。这项概念验证研究使用“瓶中基因组”（GIAB）阿什肯纳兹三人组，检查了来自单细胞的lrWGS数据，用于小变异（单核苷酸变异（SNV）和插入缺失）和结构变异（SV）的检测以及单倍型分型。对后代的单细胞（1个细胞）和多细胞（10个细胞）样本进行了lrWGS。通过利用长读长和家系信息获得了染色体长度的单倍型。通过用匹配的亲本单倍型替换这些单倍型，进一步对其进行了优化。在单细胞和多细胞样本中，92%和98%的杂合SNV以及74%和78%的杂合插入缺失被准确地进行了单倍型分型。应用于人类胚胎的植入前基因检测（PGT）时，lrWGS在检测单基因疾病方面与基于芯片的方法显示出100%的一致性，且无需定相参考。非整倍体被准确检测到，并从亲本独特等位基因分数（UAF）推断出染色体异常的机制起源。我们表明，基于lrWGS的单细胞并发单倍型分型和非整倍体分析为当前的PGT方法提供了一种替代方案，在基于细胞的产前诊断以及动植物育种等领域具有应用潜力。
