Abstract
Large variation exists in mitochondrial DNA (mtDNA) not only between but also within individuals. Also in human cancer, tumor-specific mtDNA variation exists. In this work, we describe the comparison of four methods to extract mtDNA as pure as possible from frozen tumor tissue. Also, three state-of-the-art methods for sensitive detection of mtDNA variants were evaluated. The main aim was to develop a procedure to detect low-frequent single-nucleotide mtDNA-specific variants in frozen tumor tissue. We show that of the methods evaluated, DNA extracted from cytosol fractions following exonuclease treatment results in highest mtDNA yield and purity from frozen tumor tissue (270-fold mtDNA enrichment). Next, we demonstrate the sensitivity of detection of low-frequent single-nucleotide mtDNA variants (≤1% allele frequency) in breast cancer cell lines MDA-MB-231 and MCF-7 by single-molecule real-time (SMRT) sequencing, UltraSEEK chemistry based mass spectrometry, and digital PCR. We also show de novo detection and allelic phasing of variants by SMRT sequencing. We conclude that our sensitive procedure to detect low-frequent single-nucleotide mtDNA variants from frozen tumor tissue is based on extraction of DNA from cytosol fractions followed by exonuclease treatment to obtain high mtDNA purity, and subsequent SMRT sequencing for (de novo) detection and allelic phasing of variants.
Highlights
The past decades, extensive genomic analysis of tumor specimens using massive parallel sequencing by large sequencing consortia have revealed the major somatic drivers of human cancer, that have been reported in numerous studies
We aimed to develop a sensitive procedure to detect low-frequent single-nucleotide mitochondrial DNA (mtDNA) variants from frozen tumor tissue
We started by establishing an extraction procedure to obtain mtDNA as pure as possible from frozen tumor tissue
Summary
The past decades, extensive genomic analysis of tumor specimens using massive parallel sequencing by large sequencing consortia (e.g. https://www.icgc.org/icgc and http://cancergenome.nih.gov/) have revealed the major somatic drivers of human cancer, that have been reported in numerous studies. There are fixed NUMTs present in virtually every human genome–and reported in the human reference genome–inserted millions of years ago, and more recent NUMT insertions have been described[19] Due to their sequence similarity to mtDNA, NUMTs can interfere with accurate variant detection and investigation of mitochondrial heteroplasmy[16,19,20,21,22,23]. In tumor cells the processes shaping nDNA25,26 are substantially different from the one that shapes the mtDNA13, resulting in somatic variants in NUMTs and complicating accurate mtDNA heteroplasmy detection even further for tumor cells. In the study described here, we aimed to develop a sensitive procedure to detect low-frequent single-nucleotide mtDNA variants in frozen tumor tissue. We evaluated three state-of-the-art techniques for the detection of low-frequent mtDNA-specific variants: Pacific Biosciences’ SMRT sequencing[35], UltraSEEK chemistry[36] and digital PCR
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