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Simultaneous detection of target CNVs and SNVs of thalassemia by multiplex PCR and next‑generation sequencing.

Thalassemia is caused by complex mechanisms, including copy number variants (CNVs) and single nucleotide variants (SNVs). The CNV types of α‑thalassemia are typically detected by gap‑polymerase chain reaction (PCR). The SNV types are detected by Sanger sequencing. In the present study, a novel method was developed that simultaneously detects CNVs and SNVs by multiplex PCR and next‑generation sequencing (NGS). To detect CNVs, 33 normal samples were used as a cluster of control values to build a baseline, and the A, B, C, and D ratios were developed to evaluate‑SEA, ‑α4.2, ‑α3.7, and compound or homozygous CNVs, respectively. To detect other SNVs, sequencing data were analyzed using the system's software and annotated using Annovar software. In a test of performance, 128 patients with thalassemia were detected using the method developed and were confirmed by Sanger sequencing and gap‑PCR. Four different CNV types were clearly distinguished by the developed algorithm, with ‑SEA, ‑α3.7, ‑α4.2, and compound or homozygous deletions. The sensitivities for each CNV type were 96.72% (59/61), 97.37% (37/38), 83.33% (10/12) and 95% (19/20), and the specificities were 93.94% (32/33), 93.94% (32/33), 100% (33/33) and 100% (33/33), respectively. The SNVs detected were consistent with those of the Sanger sequencing.