Sequencing Mitochondrial Transcriptomes with RNA-Seq

Thousands of mitochondrial genomes have been sequenced, but there are comparatively few available mitochondrial transcriptomes. This might soon be changing. High-throughput RNA sequencing (RNA-Seq) techniques have made it fast and cheap to generate massive amounts of mitochondrial transcriptomic data. Here, we explore the utility of RNA-Seq for assembling mitochondrial genomes and studying their expression patterns.

Specifically, researchers from the University of Western Ontario investigate the mitochondrial transcriptomes from Polytomella non-photosynthetic green algae, which have among the smallest, most reduced mitochondrial genomes from the Archaeplastida as well as fragmented rRNA-coding regions, palindromic genes, and linear chromosomes with telomeres. Isolation of whole genomic RNA from the four known Polytomella species followed by Illumina paired-end sequencing generated enough mitochondrial-derived reads to easily recover almost-entire mitochondrial genome sequences. Read-mapping and coverage statistics also gave insights into Polytomella mitochondrial transcriptional architecture, revealing polycistronic transcripts and the expression of telomeres and palindromic genes.

Polytomella mitochondrial transcription.


 A. Mitochondrial genome maps of the four known Polytomella lineages. All four genomes are made up of linear chromosomes (chr) with terminal inverted repeat (TIR) telomeres and contain 10 unique genes, including the small- and large-subunit rRNA (SSU and LSU) genes, which are fragmented and scrambled into 4 and 8 loci, respectively. The P. magna mtDNA contains 10 palindromic repeats (boxed in dark or light gray, and labeled with black circles), which contain putative functional (green) and putative nonfunctional gene copies (white). Regions represented in the RNA-Seq data are highlighted in blue. Mitochondrial contigs identified from de novo transcriptome assemblies are shown with solid lines. B. Tree of Polytomella algae and Chlamydomonas reinhardtii, based on phylogenetic analysis of Smith et al. (2013). C. Transcription of single-stranded (ss) hairpin-loop telomeres from P. capuana. D. Log-scale RNA-Seq coverage of Polytomella mitochondrial chromosomes: log(coverage + 1)/log(maximum coverage + 1).

A major goal of this study was to evaluate the utility of RNA-Seq for recovering mitochondrial genome sequences. On this front, the researchers were successful: moderate amounts of RNA-Seq data from Polytomella species easily gave near-complete mitochondrial genome assemblies. The extremely small sizes and polycistronic transcriptional organizations of Polytomella mitochondrial chromosomes undoubtedly facilitated their efficient recovery from the RNA data. Nonetheless, they believe that the approach employed here can be used to generate organelle genome sequences from other eukaryotic species, including those with larger mtDNAs than Polytomella spp.

Ultimately, RNA-Seq is a promising, cost-effective technique for studying mitochondrial genetics, but it does have drawbacks. One of its greatest potentials, as shown here, is that it can be used to generate near-complete mitochondrial genome sequences, which could be particularly useful in situations where there is a lack of available mtDNA data.

Tian Y, Smith DR. (2016) Recovering complete mitochondrial genome sequences from RNA-Seq: A case study of Polytomella non-photosynthetic green algae. Mol Phylogenet Evol [Epub ahead of print]. [article]

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