Simultaneous detection of target CNVs and SNVs of thalassemia by multiplex PCR and next­generation sequencing.

Thalassemia is caused by complex mechanisms, including copy number variants (CNVs) and single nucleotide variants (SNVs). The CNV types of α­thalassemia are typically detected by gap­polymerase 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 next­generation 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 evaluate­SEA, ­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 gap­PCR. 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.