Ultra-deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in Chinese hamster ovary cells

Abstract

Recent sequencing of the Chinese hamster ovary (CHO) cell and Chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering.

Genetic and Epigenetic Variation across Genes Involved in Energy Metabolism and Mitochondria of Chinese Hamster Ovary Cell Lines

“…Ensuring minimal influence from nuclear reads during mitochondrial genome analysis, whole‐genome sequencing reads were mapped to the Chinese hamster reference genome assembly including nuclear scaffolds (criGri1.0) and the mitochondrial genome (KX576660) . Genome alignment was performed using BWA vers.0.7.13 mem algorithm followed by the removal of polymerase chain reaction (PCR) duplicates with Picard tools vers.2.3.0 .…”

“…Partridge et al released the mitochondrial genome (mtDNA) for a CHO A L hybrid cell line and compared it to other published hamster, mouse, and rat mtDNA sequences, reporting interesting regions of high and low conservation. Recently, high‐resolution sequencing of mtDNA from 22 CHO cell lines (ATCC CHO‐K1, ATCC CHO‐S, ATCC DG44, 12 industrial, and 5 laboratory cell lines) reported mutations that were detected even at very low frequency, demonstrating cell line‐specific as well as widespread heteroplasmy …”

CHO‐Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction

“…In addition to the functional relevance, mitochondrial DNA (mtDNA) itself has several characteristics that make it a desirable investigative target. First, compared to the potentially overwhelming size of the nuclear genome, analysis of the mitochondrial genome is more approachable at approximately 16 kb in length containing 37 genes (13 proteins, 22 tRNAs, and 2 ribosomal RNAs) each lacking introns with little or no intergenic sequence . Second, mtDNA a 5‐15‐fold higher mutation rate compared to the nuclear genome due to inefficient DNA repair mechanisms and proximity of the mitochondrial genome to reactive oxygen species .…”

“…Second, mtDNA a 5‐15‐fold higher mutation rate compared to the nuclear genome due to inefficient DNA repair mechanisms and proximity of the mitochondrial genome to reactive oxygen species . Finally, mitochondrial DNA is easily enriched making it amenable to ultra‐deep NGS . Individual cells may contain hundreds to thousands of mitochondria resulting in mixtures of wild‐type and mutated mitochondrial genomes .…”

“…In total, 197 homoplasmic and heteroplasmic variants were identified across the 22 CHO cell lines. SNP or Indel mutations were identified in all protein‐coding genes, 8 tRNAs, and both rRNA genes . While the heteroplasmic nature of mitochondrial DNA poses challenges to understanding their effect on bioproduction, this knowledge may lead to novel approaches in cell engineering and screening techniques.…”