title: 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.
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Kelly
authors.affiliation: National Institute for Bioprocessing Research and Training, Fosters Avenue, Blackrock, Co., Dublin, Ireland.
authors.forename: Colin
authors.initials: C
authors.lastname: Clarke
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Alan
authors.initials: A
authors.lastname: Costello
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; National Institute for Bioprocessing Research and Training, Fosters Avenue, Blackrock, Co., Dublin, Ireland.
authors.forename: Craig
authors.initials: C
authors.lastname: Monger
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Justine
authors.initials: J
authors.lastname: Meiller
authors.affiliation: University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, Vienna 1190, Austria.
authors.forename: Heena
authors.initials: H
authors.lastname: Dhiman
authors.affiliation: University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, Vienna 1190, Austria.
authors.forename: Nicole
authors.initials: N
authors.lastname: Borth
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Martin
authors.initials: M
authors.lastname: Clynes
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address: niall.barron@dcu.ie.
authors.forename: Niall
authors.initials: N
authors.lastname: Barron
mesh_terms: D000483:Alleles; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005787:Gene Frequency; D054629:Genome, Mitochondrial; D059014:High-Throughput Nucleotide Sequencing; D054643:INDEL Mutation; D020641:Polymorphism, Single Nucleotide
keywords: Biopharmaceutical; Chinese hamster ovary; Genomics; Heteroplasmy; Mitochondrial; Next generation sequencing