{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:29:27Z","timestamp":1785414567468,"version":"3.56.0"},"reference-count":32,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Subjects of Maternal and Child Health Care in Jiangsu Province\u2014Prenatal Diagnosis","award":["FXK201747"],"award-info":[{"award-number":["FXK201747"]}]},{"name":"Scientific research project of Jiangsu Province Association of Maternal and Child Health","award":["FYX201903"],"award-info":[{"award-number":["FYX201903"]}]},{"name":"Jiangsu Province Center for Innovation in Obstetrics and Gynecology","award":["CXZX202229"],"award-info":[{"award-number":["CXZX202229"]}]},{"award":["CXZX202229"],"award-info":[{"award-number":["CXZX202229"]}],"id":[{"id":"https:\/\/ror.org\/04c1v2b17","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background\/Objectives: Non-invasive prenatal testing for monogenic disorders (NIPT-MD) provides a safe alternative to invasive procedures. However, its clinical utility is often limited by challenges such as low fetal fractions (FF), technical artifacts, and the difficulty in resolving maternally inherited pathogenic variants from the overwhelming background of maternal cell-free DNA (cfDNA). This study aimed to develop and validate a robust NIPT-MD platform for monogenic disorders, applicable across major common Mendelian inheritance patterns, by enhancing the accuracy of fetal variant detection in cfDNA. Methods: We developed a novel NIPT-MD platform validated using both simulated samples and a retrospective clinical cohort. The workflow involves a single-strand capture library preparation incorporating unique molecular identifiers (UMIs) to mitigate amplification artifacts. FF was precisely quantified using a fixed panel of high-minor-allele-frequency single-nucleotide polymorphisms (SNPs). Fetal genotypes were then inferred by resolving parental haplotypes through a statistical model that integrates weighting of variant allele frequency (VAF) and haplotype-informative SNP counts. Results: The single-strand capture protocol incorporating UMIs significantly reduced amplification biases. Validation of the fetal DNA fraction estimation algorithm revealed strong concordance with a Y-chromosome-derived method and expected spike-in samples. Optimization of the FF estimation panel conferred greater experimental stability. The platform reliably detected both paternally and maternally inherited pathogenic variants. In a retrospective cohort of 35 clinical cases, the NIPT-MD platform achieved high concordance with genotypes determined by invasive testing. Conclusions: These findings present an accurate and robust NIPT-MD platform for monogenic disorders, with high concordance to invasive testing validated in a retrospective cohort of 35 clinical cases. This method holds promise for clinical application in managing families at high risk of monogenic diseases.<\/jats:p>","DOI":"10.3390\/diagnostics16152344","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T08:15:56Z","timestamp":1785140156000},"page":"2344","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated Single-Strand DNA Capture and Haplotype-Guided Variant Calling Platform for Non-Invasive Prenatal Testing of Monogenic Disorders"],"prefix":"10.3390","volume":"16","author":[{"given":"Jie","family":"Shen","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China"},{"name":"National Engineering Laboratory for Key Technology of Birth Defect Control and Prevention, Screening and Diagnostic R and D Center, Hangzhou 310020, China"},{"name":"Biosan Clinical Laboratory, Hangzhou 310020, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7728-7615","authenticated-orcid":false,"given":"Wei","family":"Liu","sequence":"additional","affiliation":[{"name":"Center for Obstetrics and Gynecology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Lu","sequence":"additional","affiliation":[{"name":"National Engineering Laboratory for Key Technology of Birth Defect Control and Prevention, Screening and Diagnostic R and D Center, Hangzhou 310020, China"},{"name":"Biosan Clinical Laboratory, Hangzhou 310020, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Yu","sequence":"additional","affiliation":[{"name":"National Engineering Laboratory for Key Technology of Birth Defect Control and Prevention, Screening and Diagnostic R and D Center, Hangzhou 310020, China"},{"name":"Biosan Clinical Laboratory, Hangzhou 310020, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0159-6048","authenticated-orcid":false,"given":"Yin","family":"Wang","sequence":"additional","affiliation":[{"name":"National Engineering Laboratory for Key Technology of Birth Defect Control and Prevention, Screening and Diagnostic R and D Center, Hangzhou 310020, China"},{"name":"Biosan Clinical Laboratory, Hangzhou 310020, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ningyuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China"},{"name":"Center for Molecular Reproductive Medicine, Nanjing University, Nanjing 210008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Lin","sequence":"additional","affiliation":[{"name":"Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China"},{"name":"Center for Molecular Reproductive Medicine, Nanjing University, Nanjing 210008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenyu","family":"Diao","sequence":"additional","affiliation":[{"name":"Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China"},{"name":"Center for Molecular Reproductive Medicine, Nanjing University, Nanjing 210008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huijun","family":"Li","sequence":"additional","affiliation":[{"name":"Center for Obstetrics and Gynecology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Xiao","sequence":"additional","affiliation":[{"name":"National Engineering Laboratory for Key Technology of Birth Defect Control and Prevention, Screening and Diagnostic R and D Center, Hangzhou 310020, China"},{"name":"Biosan Clinical Laboratory, Hangzhou 310020, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2335-1076","authenticated-orcid":false,"given":"Xiangyu","family":"Zhu","sequence":"additional","affiliation":[{"name":"Center for Obstetrics and Gynecology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2613-3165","authenticated-orcid":false,"given":"Jun","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1007\/s12687-018-0376-2","article-title":"Rare single gene disorders: Estimating baseline prevalence and outcomes worldwide","volume":"9","author":"Blencowe","year":"2018","journal-title":"J. Community Genet."},{"key":"ref_2","first-page":"1015","article-title":"Carrier screening for 223 monogenic diseases in Chinese population: A multi-center study in 33 104 individuals","volume":"44","author":"Hou","year":"2024","journal-title":"Nan Fang Yi Ke Da Xue Xue Bao"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1002\/uog.20353","article-title":"Risk of miscarriage following amniocentesis or chorionic villus sampling: Systematic review of literature and updated meta-analysis","volume":"54","author":"Salomon","year":"2019","journal-title":"Ultrasound Obstet. Gynecol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/S0140-6736(97)02174-0","article-title":"Presence of fetal DNA in maternal plasma and serum","volume":"350","author":"Lo","year":"1997","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"61ra91","DOI":"10.1126\/scitranslmed.3001720","article-title":"Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus","volume":"2","author":"Lo","year":"2010","journal-title":"Sci. Transl. Med."},{"key":"ref_6","first-page":"578","article-title":"DNA sequencing versus standard prenatal aneuploidy screening","volume":"371","author":"Bianchi","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1734","DOI":"10.1056\/NEJM199812103392402","article-title":"Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma","volume":"339","author":"Lo","year":"1998","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2014","DOI":"10.1056\/NEJMc2216144","article-title":"High-Resolution and Noninvasive Fetal Exome Screening","volume":"389","author":"Brand","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1186\/s13073-021-00836-8","article-title":"Noninvasive prenatal testing of alpha-thalassemia and beta-thalassemia through population-based parental haplotyping","volume":"13","author":"Chen","year":"2021","journal-title":"Genome Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"673.e1","DOI":"10.1016\/j.ajog.2025.06.043","article-title":"Expanded noninvasive prenatal screening for dominant single-gene disorders: Proof-of-concept, performance, and challenges","volume":"233","author":"Chen","year":"2025","journal-title":"Am. J. Obstet. Gynecol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s13073-025-01588-5","article-title":"Universal noninvasive prenatal diagnosis for monogenic disorders using cell-free plasma DNA","volume":"18","author":"Zhang","year":"2025","journal-title":"Genome Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1159\/000528033","article-title":"Noninvasive Prenatal Diagnosis of Beta-Thalassemia Disease by Using Digital PCR Analysis of Cell-Free Fetal DNA in Maternal Plasma","volume":"49","author":"Charoenkwan","year":"2022","journal-title":"Fetal Diagn. Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1515\/cclm-2017-0689","article-title":"Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: Detection of NF1 and CFTR mutations using droplet digital PCR","volume":"56","author":"Gruber","year":"2018","journal-title":"Clin. Chem. Lab Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"958","DOI":"10.1093\/clinchem\/hvaa104","article-title":"Noninvasive Fetal Genotyping by Droplet Digital PCR to Identify Maternally Inherited Monogenic Diabetes Variants","volume":"66","author":"Caswell","year":"2020","journal-title":"Clin. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1373\/clinchem.2016.268375","article-title":"Universal Haplotype-Based Noninvasive Prenatal Testing for Single Gene Diseases","volume":"63","author":"Hui","year":"2017","journal-title":"Clin. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Li, J., Lu, J., Su, F., Yang, J., Ju, J., Lin, Y., Xu, J., Qi, Y., Hou, Y., and Wu, J. (2022). Non-Invasive Prenatal Diagnosis of Monogenic Disorders Through Bayesian- and Haplotype-Based Prediction of Fetal Genotype. Front. Genet., 13.","DOI":"10.3389\/fgene.2022.911369"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1038\/s41421-022-00457-4","article-title":"Genetic deconvolution of fetal and maternal cell-free DNA in maternal plasma enables next-generation non-invasive prenatal screening","volume":"8","author":"Xu","year":"2022","journal-title":"Cell Discov."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1093\/qjmed\/hcaf017","article-title":"Non-invasive prenatal testing for dominant single-gene disorders using targeted next-generation sequencing","volume":"118","author":"Zhang","year":"2025","journal-title":"QJM Int. J. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1093\/clinchem\/hvae189","article-title":"Routine Prenatal cfDNA Screening for Autosomal Dominant Single-Gene Conditions","volume":"71","author":"Adams","year":"2025","journal-title":"Clin. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1097\/GRF.0000000000000795","article-title":"Noninvasive Prenatal Screening for Single-Gene Disorders","volume":"66","author":"Gonsalves","year":"2023","journal-title":"Clin. Obstet. Gynecol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1016\/j.ejogrb.2020.08.001","article-title":"Non-invasive prenatal diagnosis and screening for monogenic disorders","volume":"253","author":"Scotchman","year":"2020","journal-title":"Eur. J. Obstet. Gynecol. Reprod. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1016\/j.ajhg.2017.07.012","article-title":"Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping","volume":"101","author":"Vermeulen","year":"2017","journal-title":"Am. J. Hum. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1038\/ejhg.2016.195","article-title":"Non-invasive prenatal diagnosis of spinal muscular atrophy by relative haplotype dosage","volume":"25","author":"Parks","year":"2017","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1002\/pd.4781","article-title":"Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype dosage","volume":"36","author":"Parks","year":"2016","journal-title":"Prenat. Diagn."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"14382","DOI":"10.1038\/s41598-019-50378-8","article-title":"A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT","volume":"9","author":"Tsao","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1373\/clinchem.2004.042713","article-title":"Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation","volume":"51","author":"Birch","year":"2005","journal-title":"Clin. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1664","DOI":"10.1373\/clinchem.2008.111385","article-title":"Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma","volume":"54","author":"Lun","year":"2008","journal-title":"Clin. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1038\/s41525-021-00247-z","article-title":"Estimation of cell-free fetal DNA fraction from maternal plasma based on linkage disequilibrium information","volume":"6","author":"Ju","year":"2021","journal-title":"npj Genom. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.1373\/clinchem.2007.092734","article-title":"The origin of circulating free DNA","volume":"53","author":"Pretorius","year":"2007","journal-title":"Clin. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Chandrananda, D., Thorne, N.P., and Bahlo, M. (2015). High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA. BMC Med. Genom., 8.","DOI":"10.1186\/s12920-015-0107-z"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1373\/clinchem.2011.169318","article-title":"Nonhematopoietically derived DNA is shorter than hematopoietically derived DNA in plasma: A transplantation model","volume":"58","author":"Zheng","year":"2012","journal-title":"Clin. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"41","DOI":"10.47102\/annals-acadmedsg.2022409","article-title":"Case studies of fetal mosaicisms detected by non-invasive prenatal testing","volume":"52","author":"Gosavi","year":"2023","journal-title":"Ann. Acad. Med. Singap."}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/15\/2344\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T08:25:21Z","timestamp":1785140721000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/15\/2344"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,27]]},"references-count":32,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["diagnostics16152344"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics16152344","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,27]]}}}