An evaluation of mitochondrial DNA resequencing array mitochip version 2.0 in comparison with sanger sequencing

Mitochondrial disorders can be caused by defects in mitochondrial DNA (mtDNA) or nuclear DNA. The mitochondrial genome is a small double stranded circular DNA of 16,569 base pairs. Mitochondrial DNA insertions, deletions, and hundreds of point mutations have been reported to be associated with mitochondrial disorders. The mutations that occur in the mitochondrial DNA are the main reason for mitochondrial disorders, so it is important to develop a fast, accurate, and cost effective method to screen mtDNA variances. The GeneChip® Human Mitochondrial Resequencing Array Version 2.0 (MitoChip v2.0) from Affymetrix is designed to resequence the whole mitochondrial DNA genome with high call rate (% of the bases the software was able to identify) and accuracy. The goal of this study is to evaluate MitoChip v2.0 performance by comparing it with the sequencing "gold standard" method, Sanger dideoxy sequencing, to determine the clinical utility of the MicoChip v2.0. Eleven mtDNA samples were resequenced using the MitoChip. Thirty-eight primer pairs have been designed to sequence mitochondrial genomes using ARUP's standard protocol. Seven of the samples were from patients with known well-characterized mutations or deletion. The other four samples did not have known mitochondrial DNA mutations. The present study showed an average call rate of 95.54% of the bases sequenced, at a quality score of 3; the average iv accuracy of the bases the MitoChip was able to call was 99.96%. Almost half (44.54%) of the bases called were in regions with four cytosines or more. There were some heavily clustered no call bases regions and some bases yielded "no calls" for all the samples. The present study showed a variant detection accuracy of 94.73% of the called bases. All the missing variants were due to no calls. The study did show a sensitivity level of detection of 20% heteroplasmy or greater, but the false positive rate for heteroplasmy was very high. Overall, MitoChip is a high throughput method of resequencing the mitochondrial DNA genome with reasonable accuracy and heteroplasmy level detection sensitivity. It is cost effective and easy to use, but an independent method might be needed to confirm variants; mutations could be missed due to no call bases; the heteroplasmy calls are not reliable. These issues make MitoChip v2.0 preferable as a screening tool rather than as a confirmatory test.