An oligonucleotide microarray for high-throughput sequencing of the mitochondrial genome.
Previously we developed an oligonucleotide sequencing microarray (MitoChip) as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The first generation MitoChip, however, was not tiled with probes for the noncoding D-loop region, a site frequently mutated in human cancers. Here we report the development of a second-generation MitoChip (v2.0) with oligonucleotide probes to sequence the entire mitochondrial genome. In addition, the MitoChip v2.0 contains redundant tiling of sequences for 500 of the most common haplotypes including single-nucleotide changes, insertions, and deletions. Sequencing results from 14 primary head and neck tumor tissues demonstrated that the v2.0 MitoChips detected a larger number of variants than the original version. Multiple coding region variants detected only in the second generation MitoChips, but not the earlier chip version, were further confirmed with conventional sequencing. Moreover, 31 variations in noncoding region were identified using MitoChips v2.0. Replicate experiments demonstrated >99.99% reproducibility in the second generation MitoChip. In seven head and neck cancer samples with matched lymphocyte DNA, the MitoChip v2.0 detected at least one cancer-associated mitochondrial mutation in four (57%) samples. These results indicate that the second generation MitoChip is a high-throughput platform for identification of mitochondrial DNA mutations in primary tumors.
Zhou
Shaoyu
S
Department of Otolaryngology and Head and Neck Surgery, Johns Hopkins University School of Medicine, 1550 Orleans St. North, Baltimore, MD 21231, USA.
Kassauei
Keyaunoosh
K
Cutler
David J
DJ
Kennedy
Giulia C
GC
Sidransky
David
D
Maitra
Anirban
A
Califano
Joseph
J
D001483:Base Sequence; D015331:Cohort Studies; D004272:DNA, Mitochondrial; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results
D004272:DNA, Mitochondrial