Abstract:

Objective To evaluate the performance of next-generation sequencing for genetic testing of inherited diseases in external quality assessment（EQA） program and improve the quality of detection results. Methods In 2024 EQA program，3 different neurogenetic disorders including Prader-Willi syndrome（PWS），Duchenne muscular dystrophy（DMD） and Huntington's disease（HD） were investigated. Totally，2 types of genetic variations of copy number variations（CNV） and dynamic mutations were involved. The sample panel consisted of 3 different cell cultures. Participating laboratories were asked to report the results before deadlines. The overall coincidence of detection and report of the pathogenic variations were calculated，and the basic next-generation sequencing conditions，wet-lab procedure，bioinformatics analysis workflow，quality control measures were also analyzed. Results A total of 16 laboratories enrolled in the EQA program and 11 valid laboratory results were received in the EQA program. The coincidence rate of CNV and dynamic mutations were 100.0%（22/22） and 81.8%（9/11），respectively. The overall coincidence rates were 93.9%（31/33）. Among the 11 laboratories that returned valid results，8（72.7%） completed both "wet lab" and "dry lab" procedure independently，while the other 3 laboratories had different levels of testing outsourcing. The 9（81.8%） employed whole-exome sequencing（WES），and 2（19.2%） used whole-genome sequencing（WGS）. The detection scope of all 11 laboratories covered single nucleotide variations（SNV），small insertions-deletion（InDel），as well as CNV，but varied in CNV detection range. The overall quality of sequencing data for all the 33 samples was high，with the proportion of sequencing data meeting the Q30 standard ranging from 91.60% to 96.91%. The probe capture efficiency of the 27 samples for WES varied（49.20%-87.91%）. All the 11 laboratories specified the minimum concentration of sequencing libraries，but the quality control requirements for other steps varied. Conclusions Some laboratories in Shanghai need to enhance their detection capabilities for specific mutation types. There is a need for additional improvements in the experimental procedure as well as quality control of NGS applications for detecting genetic variations.

Key words: Inherited disease, Genetic variation, Next-generation sequencing, External quality assessment, Shanghai