Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations

Abstract

BackgroundDetecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected.ResultsHere we use 20 previously characterized Sanger-sequenced positive controls in disease-causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples.ConclusionsWe have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success.

Computational Errors and Biases in Short Read Next Generation Sequencing

“…An important variant calling application is a clinical testing: Finding disease-associated mutations. Variant calling from NGS can help to detect mutations with a lower frequency than traditionally used Sanger sequencing [159].…”

“…• A SV (inversions, translocations, or large Indels) detection tools are BreakDancer [180], Breakpointer [181], CLEVER [182], SVMerge [159].…”

“…To overcome this problem, a set of QC metrics to increase reproducibility between SNV-calling pipelines was developed by Wong et al [159]. These metrics depend only on reference genome used in the alignment without accessing the raw and intermediate data or knowing the SNV calling details.…”

Computational problems of analysis of short next generation sequencing reads

“…A valuable variant calling application is clinical testing: identifying disease-associated mutations (Chin et al, 2013).…”

Identification of a Novel Nonsense Mutation in the ARSE Gene of a Patient with X-Linked Recessive Chondrodysplasia Punctata

“…The advantages of NGS its wide genome coverage and the ability to obtain from a single DNA strand. In a study on the clinical analytical sensitivity and specificity of NGS, the sensitivity was found to be 92.7% 12) . Therefore, in the cases of many other Mendelian genetic disorders that have many possible candidate genes, we recommend NGS.…”