GSE124420
Next generation sequencing reveals no change in mtDNA deletions upon the loss of mitochondrial fusion in mouse hearts
Aim: To determine whether mtDNA deletions arise upon the loss of mitochondria fusion in heart tissue Method: DNA was isolated heart mitochondria from control and heart-specific double mitofusin1-2 (dMfn) KO animals and used to generate libraries for sequencing to detect mtDNA deletions. Total genomic DNA from the Deletor mouse was provided by Anu Suomalainen-Wartiovaara and used as a positive control for the detection of mtDNA rearrangements. A standard Illumina TrueSeq paired-end library was prepared with ~500 base pair fragment inserts. Paired-end 100 base pair sequencing was conducted using an Illumina HiSeq 2500. The reads were mapped to the genomic sequence without the mitochondria using bowtie (VN: 2.1.0) to remove nuclear-genomic sequences. The unmapped reads were then mapped to the mitochondrial sequence (GenBank JF286601.1) using bwa (n=0.04, VN: 0.6.2-r126)  with unmapped reads undergoing an additional mapping round after trimming fastx_trimmer, VN: 0.0.13.2) to ensure higher mapping results. Using samtools (VN: 1.0: samtools –f1 –F14) reads, where the two paired sequences were observed to be greater than 600 base pair apart were identified as those containing deletion breakpoints. These reads were extracted for additional analysis. Results: Control and dMfn KO samples showed similar frequencys of breakpoints, while the positive control (Deletor mouse) showed a high frequecny of large breakpoints. Overall design: Heart DNA from littermate control (3 replicates), dMfn KO (3 replicates), Deletor mouse as positive control (1 replicate)