NGS4THAL, a one-stop molecular diagnosis and carrier screening tool for thalassemia and other hemoglobinopathies by next-generation sequencing

Background Thalassemia is one of the most common genetic diseases and a major health threat worldwide. Accurate, efficient and scalable genetic testing methodology is much needed for its molecular diagnosis and carrier screening.Results We developed NGS4THAL, a bioinformatics analysis pipeline analyzing next generation sequencing (NGS) data to detect pathogenic variants for thalassemia and other hemoglobinopathies. NGS4THAL recovers and realigns ambiguously mapped NGS reads derived from the homologous hemoglobin gene clusters to achieve accurate detection of point mutations and small insertion/deletions (InDels). And it uses several structural variant (SV) detection tools with complementary algorithms, and an inhouse database with control data on a number of known SVs to achieve accurate detection of hemoglobin SVs. Detected variants are matched with those in HbVar, allowing recognition of known pathogenic variants, including disease modifiers. Tested on simulation data, NGS4THAL achieved high sensitivity and specificity. For targeted NGS sequencing data from samples with laboratory-confirmed pathogenic hemoglobin variants, it achieved 100% detection accuracy. Application of NGS4THAL on whole genome sequencing data from unrelated studies detected thalassemia mutation carrier rates for Hong Kong Chinese and Northern Vietnamese that were consistent with those from epidemiological studies.Conclusions NGS4THAL is a highly accurate and efficient molecular diagnosis tool for thalassemia and other hemoglobinopathies based on tailored analysis of NGS data, and is potentially scalable for carrier screening purposes.