J Biotechnol. 2019 Jun 20;299:8-12. doi: 10.1016/j.jbiotec.2019.04.019.

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

Strieskova L1, Gazdaricova I2, Kajsik M3, Soltys K4, Budis J5, Pos O6, Lickova M7, Klempa B7, Szemes T8.

Author

 

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 genome contains 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.

Summary

Mitochondrial genome is a compact molecule without introns [1]. The mammalian mitochondrial genome contains 37 genes encoding 22 transfer RNAs, 2 ribosomal RNAs and 13 polypeptide of oxidative phosphorylation system [2]. Since mtDNA represents only a small part of total cellular DNA, analysis of mitochondrial genome is problematic. However, diagnosis of defects in mitochondrial genome is very important, because it can alter synthesis of mitochondrially encoded proteins, several important biological processes [3], but also nuclear gene expression through the retrograde signaling [4]. It was shown that mutations in mtDNA cause several disorders affecting especially tissues with high energetic consumption [5]. Since the next-generation sequencing (NGS) become available, it has been introduced for the analysis of mitochondrial genome. However, the main problem is still low amount of mtDNA in total DNA samples. This led to the development of several enrichment methods [6]. To avoid these procedures, we have designed novel method for preparation of mtDNA enriched samples. Our protocol is based on ultracentrifugation and total RNA sequencing of cell lysate [6,7].

Because of differences between nuclear and mtDNA structure [8,9] and transcription machinery [10–12], there is a significant difference between nuclear DNA/RNA ratio and ratio of mtDNA/RNA. This is why more readings belongs to mtDNA in our total RNA sequencing based method compare to sequencing of the total human DNA [13,14]. We assembled the whole mtDNA sequence with average coverage sufficient for the determination of variants and detection of mitochondrial heteroplasmy [15].

 

Figure 1. Ultracentrifugation enrichment protocol followed by total RNA sequencing allows assembly of the complete mitochondrial genome. Vero E6: The kidney epithelioid cells lineage, extracted from African green monkey (Chlorocebus sabaeus), (A) were disrupted using freeze-melting protocol, what resulted in the release of nucleic acids into the extracellular space (B). The remaining parts of the lysed cells were removed by centrifugation (1000 x g for 5 min) and supernatant were used for additional purification by ultracentrifugation protocol (100 000 x g for 3 hours) (C). After ultracentrifugation, the pellet was used to isolate total cellular (and mitochondrial as well) RNA and reverse transcripted into cDNA (D). The resulting cDNA attended as a template for the preparation of the Nextera XT library, which was sequenced using a MiSeq machine (Illumina) (E).  The amount of mtDNA short reads were sufficient for de novo assembling and annotating of the complete Chlorocebus sabaeus mitochondrial genome (F).

 

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