Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

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Noninvasive prenatal testing of common aneuploidies has become routine over the past decade, but testing of monogenic disorders remains a challenge in clinical implementation. Most recent studies have inherent limitations, such as complicated procedures, a lack of versatility, and the need for prior knowledge of parental genotypes or haplotypes. To overcome these limitations, a robust and versatile next‐generation sequencing‐based cell‐free DNA (cfDNA) allelic molecule counting system termed cfDNA barcode‐enabled single‐molecule test (cfBEST) is developed for the noninvasive prenatal diagnosis (NIPD) of monogenic disorders. The accuracy of cfBEST is found to be comparable to that of droplet digital polymerase chain reaction (ddPCR) in detecting low‐abundance mutations in cfDNA. The analytical validity of cfBEST is evidenced by a β‐thalassemia assay, in which a blind validation study of 143 at‐risk pregnancies reveals a sensitivity of 99.19% and a specificity of 99.92% on allele detection. Because the validated cfBEST method can be used to detect maternal‐fetal genotype combinations in cfDNA precisely and quantitatively, it holds the potential for the NIPD of human monogenic disorders.