Next-generation sequencing (NGS) is emerging as a new method for the detection of clinically significant copy number variants (CNVs). In this study, we developed and validated rapid CNV-sequencing (rCNV-seq) for clinical application in prenatal diagnosis. Low-pass whole-genome sequencing was performed on PCR libraries prepared from amniocyte genomic DNA. From 10–40 ng of input DNA, PCR-free libraries consistently produced sequencing data with high unique read mapping ratios, low read redundancy, low coefficient of variation for all chromosomes and high genomic coverage. In validation studies, reliable and accurate CNV detection using PCR-free-based rCNV-seq was demonstrated for a range of common trisomies and sex chromosome aneuploidies as well as microdeletion and duplication syndromes. In reproducibility studies, CNV copy number and genomic intervals closely matched those defined by chromosome microarray analysis. Clinical testing of genomic DNA samples from 217 women referred for prenatal diagnosis identified eight samples (3.7%) with known chromosome disorders. We conclude that PCR-free-based rCNV-seq is a sensitive, specific, reproducible and efficient method that can be used in any NGS-based diagnostic laboratory for detection of clinically significant CNVs.

Optical genome mapping (OGM) offers high consistency in simultaneously detecting structural and copy number variants. This study aimed to retrospectively evaluate the efficacy and potential applications of OGM in preconception genetic counseling. Herein, 74 samples from 37 families were included, and their results of OGM were compared to conventional methods, namely karyotyping (KT) and chromosomal microarray analysis (CMA), which identified 27 variants across 16 positive families. Notably, OGM achieved a concordance rate of 94.7% and 100% with KT and CMA, respectively, presenting an overall concordance of 96.3%, as it missed detecting a centromeric translocation. Additionally, OGM detected two cryptic balanced translocations and a small deletion in three families that were missed by conventional methods, improving the diagnostic rate by 5.4%, along with assisting in the diagnoses of six families (16.2%) by identifying complex rearrangements and confirming cryptic translocations. The combination of KT with OGM yielded the highest diagnostic rate in all families. Overall, the findings of this study present the notable potential of OGM for its application, combined with KT per requirement, in clinical settings to improve the efficiency and accuracy of diagnoses and rapid screening of individuals seeking preconception genetic counseling.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is the second most common form of muscular dystrophy, which is characterized by a reduction in the number of D4Z4 repeats on chromosome 4q35. Prenatal diagnosis of FSHD has been challenging due to the large quantity and high-quality DNA required for Southern blot (SB) analysis. Optical genome mapping (OGM) technology has shown promise in identifying repeat contraction disorders and presents a potential tool for the prenatal diagnosis of FSHD. Methods: In this retrospective cohort study, we investigated the distribution of D4Z4 repeats in 100 unrelated healthy individuals from the Chinese Han population using peripheral blood samples and DLS labelling method. Additionally, prenatal diagnosis using OGM was performed in 12 FSHD families at Peking Union Medical College Hospital between January 2021 and December 2023. The prenatal samples included 2 amniotic cell cultures and 10 chorionic villus samples (CVS), with 9 labeled using DLS and 4 using NLRS method. Results: Among the 100 healthy controls, the distribution of D4Z4 repeats varied, with 3 individuals having borderline 10 repeat counts on 4qA, and the most frequent count being 14 units. One individual with mosaicism was also identified. In the cohort of 12 FSHD families,14 prenatal diagnoses were performed. Of these 14 cases, 4 fetuses tested positive for 4qA contraction, with repeat counts ranging from 2 to 4. In both families that underwent two rounds of prenatal diagnosis, the first diagnosis indicated the presence of FSHD, leading to pregnancy termination, while the second diagnosis confirmed the presence of healthy fetuses. The overall positive rate was 28.57%. Conclusion: Our findings demonstrate that OGM is an accurate and effective method for the prenatal diagnosis of FSHD. The application of OGM in prenatal settings could offer significant benefits to families affected by FSHD with reproductive concerns.

Optical genome mapping (OGM) is an emerging cytogenetic method for concurrently detecting structural variants (SVs) and copy number variants (CNVs). Howeve