PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

ABSTRACT: Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) anal

SUBMITTER: Gould MP 

PROVIDER: S-EPMC4619561 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature