Tissue and Plasma Mitochondrial DNA in Sepsis
Purified DNA was used to generate sequencing libraries using the KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA, USA) with 10-12 cycles of amplification as needed. Adaptor ligated DNA was purified by 1.5X SPRI beads and amplified by indexed primers. Ampure cleanup protocols were modified with a 1:1.4 ratio. To enrich mtDNA for sequencing, resulting libraries were hybridized using mtDNA specific, biotinylated, probes (myBaits, Arbor Biosciences) according to the manufacturers instructions. The library was size selected from 150 to 190 base pairs and sequencing was performed using the Illumina HiSeq 4000 Sequencing System (Illumina, San Diego, CA, USA) with the generation of 100 bp paired-end (PE) reads to a depth of approximately 5 million paired reads per sample.)