NGS analysis (mtDNA-seq) of the mitochondrial genome from Nicotiana tabacum with a deletion of the nad9 locus, line delta-nad9-c03
Nextgeneration sequencing was carried out at the Sequencing Core Facility of the Max Planck Institute for Molecular Genetics, Berlin. After initial quality control using the Bioanalyzer (Agilent), sequencing libraries were prepared from 12-90 ng of DNA per sample following the library preparation protocol of the KAPA DNA Hyper Prep Kit (Roche) for double indexed Illumina libraries. First, DNA was sheared using a Covaris S2 system (duty cycle 5%, intensity 5, 40 s run time). After end repair and A-tailing, Illumina sequencing-compatible adapters carrying unique dual indices were ligated to the fragments (NEXTFLEX Unique Dual Index Barcodes). Following bead-based clean-up steps, the libraries were amplified using 7 cycles of PCR. After a second cleanup, library quality and size was checked with qBit, the Bioanalyzer and qPCR. Sequencing was carried out in an Illumina MiSeq micro flow cell in PE150bp mode yielding between 220,000 and 300,000 fragments per sample.