Ultracentrifugation enrichment protocol followed by total RNA sequencing allows assembly of the complete mitochondrial genome

Strieskova, L.a, Gazdaricova, I.b, Kajsik, M.c, Soltys, K.b,c, Budis, J.a,c,d, Pos, O.a,b, Lickova, M.e, Klempa, B.e, Szemes, T.a,b,c

aGeneton Ltd., Bratislava, Slovakia
bDepartment of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia
cComenius University Science Park, Bratislava, Slovakia
dSlovak Centre of Scientific and Technical Information, Bratislava, Slovakia
eInstitute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia

Abstract

The mitochondrial genome is an independent genetic system in each eukaryotic cell outside the nuclear genome. Mitochondrial DNA (mtDNA) appears in high copy number within one cell, unlike nuclear DNA, which exists in two copies. But nevertheless, mtDNA represent only small part of total cellular DNA what causes problematic analysis and identification of relevant mutations. While most researchers tend to overlook it because of its small size, the mitochondrial genomecontains genes that are essential for cellular energetics and survival. Because of the increased awareness on the importance of metabolism and bioenergetics in a wide variety of human diseases, more and more mtDNA studies were performed. Mitochondrial genome research has established the connection between mtDNA and a wide variety of diseases such as cancer or neurodegenerative disorders. At the present time, several methods are known, that allow sequencing of mtDNA. However, genomic analysis is often complicated due to the low content of mtDNA compared to nuclear DNA. For this reason, we have designed a new approach to obtaining the genomic mitochondrial sequence. We chose RNA based sequencing. Since human mtDNA does not contain introns, the reconstruction of whole mitochondrial genome through RNA sequencing seems to be effective. Our method is based on total RNA sequencing coupled with simple ultracentrifugation protocol and de novo assembly. Following our protocol, we were able to assemble a complete mammalian mitochondrial genome with a length of 16,505 bp and an average coverage of 156. The method is a relatively simple and inexpensive which could help in the further research or diagnostics of mtDNA-based diseases.