Challenges of Bringing Next Generation Sequencing Technologies to Clinical Molecular Diagnostic Laboratories

Abstract

Molecular diagnosis of complex dual genome mitochondrial disorders is a challenge. It requires the identification of deleterious mutations in one of the~1,500 nuclear genes and the mitochondrial genome. If the molecular defect is in the mitochondrial genome, quantification of degree of mutation load (heteroplasmy) in affected tissues is important. Due to the extreme clinical and genetic heterogeneity, conventional sequence analysis of the candidate genes one-byone is impractical, if not impossible. The newly developed massively parallel next generation sequencing (NGS) technique, that allows simultaneous sequence analysis of multiple target genes, when appropriately validated with deep coverage and proper quality controls, can be used as an effective comprehensive diagnostic approach in CLIA certified clinical laboratories.

Application of Massively Parallel Sequencing in the Clinical Diagnostic Testing of Inherited Cardiac Conditions

“…The validation process should include an evaluation of the platform that is used and the downstream data analysis [108]. Platform validation establishes the performance of the sequencing platform and the different variants that can be detected by the assay [109]. Downstream data analysis validation establishes the software parameters required to accurately read sequence data and to detect the variants [109].…”

“…Platform validation establishes the performance of the sequencing platform and the different variants that can be detected by the assay [109]. Downstream data analysis validation establishes the software parameters required to accurately read sequence data and to detect the variants [109]. These validation steps should establish the sensitivity, specificity and limitations of a particular assay.…”

Molecular diagnosis of coenzyme Q10deficiency

“…Whole genome and exome sequencing approaches are being successfully implemented in research projects [33,34], but are not yet routine strategies in diagnosis due to high costs, long turnover times (run and analysis time) and ethical issues. On the other hand, targeted resequencing using gene panel is appearing in a clinical setting due to lower sequencing costs, shorter sequencing time, simpler data analysis and greater sensitivity per gene due to the greater coverage achieved [35]. These new approaches may be ideal for diagnosis of several disorders with broad heterogenic phenotypes and genotypes [31,32], such as the mitochondrial disorders and CoQ deficiency syndromes.…”

“…In consequence, a fraction of the coding regions could remain unsequenced. False-negative and false-positive rates are higher than in targeted panels, adding a layer of complexity and uncertainty to the diagnosis [35]. Post NGS, powerful bioinformatic tools are used to assess all the highthroughput data.…”

An Innovative Data Analysis Strategy For Accurate NGS Detection of Tumor mtDNA Mutations

“…Although NGS has been extensively applied to effectively detect mtDNA mutations, there are still several big challenges existing in data analysis of mtDNA sequencing (8). Among them, most critical consideration is to decrease the number of false-positive and false-negative mutations, which are greatly affected by mtDNA sequencing depth, especially for those with low heteroplasmy level (9).…”