Estimating relative mitochondrial DNA copy number using high throughput sequencing data.

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Accurate measurement of relative mitochondria copy number (MTCN) is crucial for understanding cellular function and disease.
• High-throughput sequencing offers potential for MTCN estimation, but validation against gold-standard methods is necessary.

Purpose of the Study:

• To evaluate computational methods for estimating relative MTCN from sequencing data.
• To compare sequencing-based MTCN estimates with a gold-standard RT-PCR assay (NovaQUANT).
• To assess the reliability of the NovaQUANT assay in cancer cell lines.

Main Methods:

• Relative MTCN was measured in 13 breast cancer cell lines using the NovaQUANT kit (RT-PCR based).
• Six computational approaches were employed to estimate relative MTCN from exome sequencing and RNA-seq data.
• Copy number variants (CNVs) of nuclear genes used in the NovaQUANT assay were analyzed in The Cancer Genome Atlas (TCGA).

Main Results:

• Relative MTCN estimated from exome sequencing data showed good correlation with RT-PCR measurements.
• RNA-seq data did not yield reliable MTCN estimates when compared to RT-PCR.
• Analysis of TCGA data revealed frequent CNVs in the nuclear genes targeted by the NovaQUANT assay in tumor cells.

Conclusions:

• Exome sequencing provides a viable method for estimating relative MTCN.
• The NovaQUANT assay's accuracy may be compromised in tumor samples due to CNVs affecting its reference nuclear genes.
• Further investigation is needed to refine gold-standard methods for MTCN quantification in cancer genomics.