An improved analytic workflow for serum mtDNA DAMP abundance, fragmentation and heteroplasmic variants: a retrospective analysis of acute respiratory failure patients

Serum mitochondrial DNA (mtDNA) fragments act as proinflammatory damage-associated molecular patterns (DAMPs), and have been linked to outcomes in critical illness. However, the prognostic value of mtDNA DAMPs remains uncertain, possibly due to confounding nuclear mitochondrial insertions (NUMTs), which obscure both quantitation and variant detection. We developed a deep sequencing and bioinformatics workflow to minimize NUMT-related artifacts. By modeling insert size distributions in ICU patients with acute respiratory failure, we excluded likely NUMT-derived fragments, improving the accuracy of mtDNA DAMP fragmentomic analysis. Short inserts (<150 bp) had low NUMT contamination, and their abundance and size correlated with mortality more strongly than total mtDNA abundance. To improve variant detection, we introduced a novel "read mismatch percentage" to identify NUMT-induced chimeras, enabling identification of mtDNA variants linked to survival and post-acute quality of life. This workflow supports the need for further studies on serum mtDNA variants to predict acute and long-term outcomes in critical illness.