The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection

Abstract

Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.

Figure 1

(A, left panel) Pictorial depiction of MitoChip hybridization data (.DAT file) after scanning in an Affymetrix Microarray suite; control tiles at the four corners of the chip permit automated grid alignment, which generates a .CEL file for subsequent batch analysis in GDAS. (right panel) Higher-magnification view of the tiling pattern on the MitoChip demonstrating the four alternative oligonucleotides (25mers with the 13th base being A, C, G, or T) for each RCRS base position, and the sequence-specific hybridization occurring at each position. All base calls are homozygous in the illustrated panel. (B) Serial dilution experiments performed with a primary lung cancer (JHU_MITO #12) and its corresponding normal DNA sample demonstrate the ability of the MitoChip to detect an aberrant clonal population in 50-fold-diluted tumor DNA. The sequence output is generated in GDAS, and the mutation detected corresponds to RCRS13197 C>T mutation in the tumor sample (Table 3). As illustrated, the mutation is detected at both positions for RCRS13197 on the MitoChip. Note that nucleotides immediately 3′ and 5′ of the mutated base position often result in a “no call” (N) due to poor hybridization quality scores caused by the mismatched base. The numbers depicted on the GDAS chromatogram correspond to the tiled base positions on the MitoChip and not the actual RCRS position. A convenient Excel-based conversion table linking the duplicate MitoChip positions to the RCRS nucleotide position is available from the authors on request. (N, normal; T, Tumor; Y, C+T in IUPAC ambiguity code)

Figure 2

Analysis of primary tumors and matched body fluid samples (urine or pancreatic juice) obtained from patients with bladder cancer (n=5) and pancreatic cancer (n=4). In each case illustrated, a heteroplasmic mitochondrial mutation identical to one seen in the primary tumor DNA (left hemi-panel) is also found in the fluid DNA (right hemi-panel). The JHU_MITO ID number, type of specimen, the mitochondrial nucleotide position involved, and the specific base change are detailed for each tumor-fluid pair illustrated.