Novel attributes of cell‐free plasma mitochondrial DNA in traumatic injury

Abstract

Dear Editor, Plasma mitochondrial DNA (mtDNA) fragment abundance has emerged as a biomarker in multiple human disorders, thus pointing to the prospect that mtDNA, like F I G U R E 1 RNA target bait-capture and bioinformatics protocol and nuclear mitochondrial (NUMT) identification. (A) DNA is isolated from plasma or tissue. In the figure, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) are denoted by colour. DNA isolation and library preparation are applied to all sample DNA, regardless of nuclear or mitochondrial origin. A target bait-capture kit consists of biotinylated RNA probes complementary to the mitochondrial genome. The probes efficiently bind mtDNA but can also bind homologous NUMT, as illustrated by the DNA fragment half coloured as mitochondrial and half coloured as nuclear. Once enriched, samples are pooled and sequenced on a standard Illumina instrument. From there, a Workflow Description Language pipeline aligns the reads to the whole genome -nuclear and mitochondrial. Three custom C/C++ programs built on the htslib library then call mitochondrial and nuclear coverage, insert (the size of a fragment after end repair and sequencing adapter ligation), and variant calling. (B) Schematic depiction of how target-bait capture also leads to the sequencing of flanking regions of polymorphic NUMTs. (C) Integrated Genome Viewer (IGV) histograms depicting a specific polymorphic NUMT in a nontransfused patient whereas the second patient lacks this insertion at either t0 or t72 post-admittance. Subfigures (A) and (B) were prepared in Inkscape.

New methods for analysing circulating cell‐free mitochondrial DNA

“…Therefore, this protocol allows for the simultaneous quantification of mtDNA levels across the entire mitochondrial genome, differentiation of fragment lengths, and the identification of mtDNA variants. 5 The authors utilised their protocol to investigate ccf-mtDNA in comparison to clinical phenotypes of injury and severity scores in a cohort of 30 trauma patients. 5 There were no significant differences in ccf-mtDNA abundance between patients with non-severe or severe injuries.…”

“…5 The authors utilised their protocol to investigate ccf-mtDNA in comparison to clinical phenotypes of injury and severity scores in a cohort of 30 trauma patients. 5 There were no significant differences in ccf-mtDNA abundance between patients with non-severe or severe injuries. The ccf-mtDNA fragment length was significantly shorter in patients with major trauma and in patients where acute lung injury occurred after admission compared to non-severe injury patients.…”

“…Therefore, the authors utilised samples from this trauma cohort to validate that their protocol can be implemented to examined mtDNA levels, fragment length, and variants. 5 This technological advance has advantages over the standard qPCR methods, as the entire mitochondrial genome can be analysed in one protocol. However, additional experiments and validation studies should be performed to directly compare the sensitivity and accuracy of this protocol versus the standard qPCR methods.…”

“…Daly and colleagues recently developed a protocol that provides a new approach to analysing plasma ccf-mtDNA. 5 In their approach, they utilised a target bait-capture kit consisting of biotinylated RNA probes complementary to the entire mitochondrial genome. First, the biotinylated probes bind to mitochondrial sequences and then are enriched, pooled, and sequenced on a standard Illumina instrument.…”

“…The authors utilised their protocol to investigate ccf‐mtDNA in comparison to clinical phenotypes of injury and severity scores in a cohort of 30 trauma patients 5 . There were no significant differences in ccf‐mtDNA abundance between patients with non‐severe or severe injuries.…”