| Autor: |
Matthew E. Kutcher, Jon D. Simmons, Hamo M, V.M. Pastukh, Hank W. Bass, Zachary M. Turpin, S.C. Groark, Mark N. Gillespie, Yong B. Tan, C.M. Francis, G. Daly, Hartsell Em, Raymond J. Langley, C.Z. Aggen, C. Edwards, Dinwiddie Dl |
| Rok vydání: |
2021 |
| Předmět: |
|
| DOI: |
10.21203/rs.3.rs-668341/v1 |
| Popis: |
The presence in plasma of mitochondrial DNA (mtDNA) fragments, a proinflammatory damage-associated molecular pattern (DAMP), is positively associated with outcomes of multiple human disorders. Because mtDNA comprises only a small percentage of the total DNA in plasma, qPCR is typically employed as an analytic strategy. However, this method provides little insight into sequence origins or other characteristics of circulating mtDNA fragments. Here we found that target bait-capture applied to plasma mtDNA derived from severely injured patients enriched recovery by ≈ 1400-fold, thus affording depth sufficient for NextGen sequence analysis. Our method excluded nuclear mitochondrial insertions (NUMTs) using a stringent alignment and filtering strategy which calls and quantifies both NUMT in the reference genome and polymorphic NUMTs. After enrichment, we sequenced 2,000–60,000x mean coverage over the mtDNA genome. Normalization of mtDNA abundance to NUMT coverage reduced batch variability. Two massively-transfused trauma patients and two non-transfused patients displayed time-dependent increases in mtDNA DAMP coverage and decreases in the mean fragment length, which were more pronounced in massively transfused patients. Finally, our approach enabled detection of low-frequency heteroplasmic variants. Collectively, these findings suggest that our target bait-capture, deep sequencing and attendant analytic protocols could provide unprecedented characterization of cell-free plasma mtDNA and NUMTs. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
